Ole J. Bjerrum

Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann's Thrombasthenia and Bernard-Soulier Syndrome

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed (125)I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two (125)I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of (125)I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.

Detection of Biospecific Interaction during the First Dimension Electrophoresis in Crossed Immunoelectrophoresis

Lectins were included in the first dimension gel in crossed immunoelectrophoresis of human serum proteins. Con A resulted in a retardation of most glycoproteins during the first electrophoresis and heterogeneous forms of individual glycoproteins could be detected. Ulex europeus lectin gave most of the proteins a higher migration velocity. Pokeweed mitogen did not interact with serum glycoproteins, but it did with serum lipoproteins. The method allows a close study of interacting components, determination of binding specificities, and detection of minor interacting components, including heterogeneous molecular forms.

Pharmacokinetics of fexofenadine: Evaluation of a microdose and assessment of absolute oral bioavailability

A human pharmacokinetic study was performed to assess the ability of a microdose to predict the pharmacokinetics of a therapeutic dose of fexofenadine and to determine its absolute oral bioavailability. Fexofenadine was chosen to represent an unmetabolized transporter substrate (P-gP and OATP). Fexofenadine was administered to 6 healthy male volunteers in a three way cross-over design. A microdose (100microg) of (14)C-drug was administered orally (period 1) and intravenously by 30min infusion (period 2). In period 3 an intravenous tracer dose (100microg) of (14)C-drug was administered simultaneously with an oral unlabelled therapeutic dose (120mg). Plasma was collected from all 3 periods and analysed for both total (14)C content and parent drug by accelerator mass spectrometry (AMS). For period 3, plasma samples were also analysed using HPLC-fluorescence to determine total drug concentration. Urine was collected and analysed for total (14)C. Good concordance between the microdose and therapeutic dose pharmacokinetics was observed. Microdose: CL 13L/h, CL(R) 4.1L/h, V(ss) 54L, t(1/2) 16h; therapeutic dose: CL 16L/h, CL(R) 6.2L/h, V(ss) 64L, t(1/2) 12h. The absolute oral bioavailability of fexofenadine was 0.35 (microdose 0.41, therapeutic dose 0.30). Despite a 1200-fold difference in dose of fexofenadine, the microdose predicted well the pharmacokinetic parameters following a therapeutic dose for this transporter dependent compound.

Crossed immunoelectrophoresis of human erythrocyte membrane proteins: Immunoprecipitation patterns for fresh and stored samples of membranes extensively solubilized with non-ionic detergents

Abstract 1. 1.|Human erythrocyte membranes (ghosts) were treated with four non-ionic detergents at pH 9.2 (5 °C) in a dilute buffer. More than 85% of the protein was solubilized. A protein concentration of up to 4 mg/ml was obtained. 2. 2.|The solubilized proteins were examined with rabbit antibodies against membrane material by crossed immunoelectrophoresis in agarose gels containing the solubilizing detergent. 3. 3.|The immunoelectrophoretic analyses showed that the membrane proteins were stable at −196 °C in the intact membrane. After solubilization with a non-ionic detergent changes occurred within two days at 5 °C but not at −20 °C. 4. 4.|A total of 19 membrane-specific immunoprecipitates were observed by crossed immunoelectrophoresis in the non-ionic detergent Berol EMU-043. Similar precipitation patterns were obtained with other non-ionic detergents. Some of the immunoprecipitates formed two peaks and some showed reaction of partial identity. 5. 5.|The antibodies precipitated most of the solubilized protein as demonstrated by immunoabsorption in an agarose gel of electrophoretically migrating antigens. 6. 6.|The precipitation pattern in crossed immunoelectrophoresis with a given antibody solution was reproducible with different membrane preparations. 7. 7.|Crossed immunoelectrophoresis in non-ionic detergents can be used for analysis of membrane protein fractions as a complement to polyacrylamide gel electrophoresis in dodecylsulfate.

Detection of amphiphilic proteins and peptides in complex mixtures. Charge-shift crossed immunoelectrophosis and two-dimensional charge-shift electrophoresis

Charge-shift electrophoresis has been suggested as a simple and novel method for differentiating between emphiphilic and hydrophilic proteins (Helenius, A. and Simons, K. (1977) Proc. Natl. Acad. Sci. U.S. 74, 529-532.) This communication reports on the combination of charge-shift electrophoresis with second dimensional quantitative immunoelectrophoresis, and on a two-dimensional modification of the charge-shift electrophoresis technique. From results obtained with unfractionated human plasma proteins and human erythrocyte membrane proteins we conclude that these modifications reliably permit detection of amphiphilic proteins and peptides in complex mixtures.

Auto- and iso-antigens of human spermatozoa detected by immunoblotting with human sera after SDS-PAGE

A sodium dodecylsulphate-polyacrylamide gel electrophoretic system for analysis of the proteins of human spermatozoa was established. Subsequent immunoblotting of the gels with human sera gave a reproducible immunolabelling of distinctive polypeptide bands. To identify auto- and isoantigens, 28 well-characterized sera from the WHO Reference Bank for Reproductive Immunology (1977, Acta Pathol. Microbiol. Scand. Sect. C, Suppl. 252) containing agglutinating and complement fixating antibodies (9 female (F) and 19 male (M) and 30 normal sera (14 F and 16 M) were analysed with reference to binding of IgG. Three spermatozoal antigens with Mr values in reduced state of 120,000 (6), 41,000 (6) and 32,000 (15) were found to be specifically correlated to the agglutinating activity of the reactive sea. (The number of these are given in parentheses). Furthermore, IgG from 2 and 3 of the normal sera and 14 and 17 of the agglutinating sera reacted with 78 and 64 kDA polypeptide, respectively. Identical binding patterns of IgG to spermatozoal polypeptides were obtained with IgG from male and female sera. The IgG-binding could not be correlated to the modes of spermatozoal agglutination. In a similar analysis of the IgM binding antigens of 21 agglutinating and 12 normal sera no differences in binding between the sera were found, except for a specific reaction to a 78 kDa antigen for 5 of the agglutinating sera.

Complement lysis: the ultrastructure and orientation of the C5b-9 complex on target sheep erythrocyte membranes.

The C5b‐9 complex derived from human serum and assembled on target sheep erythrocyte membranes is a thin‐walled cylinder rimmed by an annulus at one end. The total height of the cylinder is 150 Å, towards which the annulus contributes 30 Å. The cylinder has an apparently uniform internal diameter of 100 Å. The external diameter of the annulus is 200 Å. The classical complement ‘rings’ visualized on membranes after complement lysis represent such C5b‐9 cylinders perpendicularly oriented on the membranes. The thin‐walled cylinder is anchored in the membrane maim and the annulus located in the exterior membrane glycocalyx. At the sites of attachment of the C5b‐9 complexes, the continuity of the membrane bilayer is disturbed and the presence of trans‐membrane pores is indicated The data essentially support the ‘doughnut’ theory of complement lysis.

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.

An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations

Many antibody preparations exhibit proteolytic activity due to the presence of plasmin. In crossed immunoelectrophoresis at pH 8.6 this enzyme can degrade certain proteins during electrophoresis in the antibody‐containing gel, resulting in artefacts in the form of extra precipitation arcs of congruent shape. The degradation behaviour of spectrin, a major protein of human erythrocyte membranes, was investigated. The artefact could be completely abolished by the addition of protease inhibitors, e.g. aprotinin and soya bean trypsin inhibitor, to the antibody preparations.

Comparative pharmacokinetics between a microdose and therapeutic dose for clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen), and phenobarbital in human volunteers

A clinical study was conducted to assess the ability of a microdose (100 μg) to predict the human pharmacokinetics (PK) following a therapeutic dose of clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen) and phenobarbital, both within the study and by reference to the existing literature on these compounds and to explore the source of any nonlinearity if seen. For each drug, 6 healthy male volunteers were dosed with 100 μg (14)C-labelled compound. For clarithromycin, sumatriptan, and propafenone this labelled dose was administered alone, i.e. as a microdose, orally and intravenously (iv) and as an iv tracer dose concomitantly with an oral non-labelled therapeutic dose, in a 3-way cross over design. The oral therapeutic doses were 250, 50, and 150 mg, respectively. Paracetamol was given as the labelled microdose orally and iv using a 2-way cross over design, whereas phenobarbital was given only as the microdose orally. Plasma concentrations of total (14)C and parent drug were measured using accelerator mass spectrometry (AMS) or HPLC followed by AMS. Plasma concentrations following non-(14)C-labelled oral therapeutic doses were measured using either HPLC-electrochemical detection (clarithromycin) or HPLC-UV (sumatriptan, propafenone). For all five drugs an oral microdose predicted reasonably well the PK, including the shape of the plasma profile, following an oral therapeutic dose. For clarithromycin, sumatriptan, and propafenone, one parameter, oral bioavailability, was marginally outside of the normally acceptable 2-fold prediction interval around the mean therapeutic dose value. For clarithromycin, sumatriptan and propafenone, data obtained from an oral and iv microdose were compared within the same cohort of subjects used in the study, as well as those reported in the literature. For paracetamol (oral and iv) and phenobarbital (oral), microdose data were compared with those reported in the literature only. Where 100 μg iv (14)C-doses were given alone and with an oral non-labelled therapeutic dose, excellent accord between the PK parameters was observed indicating that the disposition kinetics of the drugs tested were unaffected by the presence of therapeutic concentrations. This observation implies that any deviation from linearity following the oral therapeutic doses occurs during the absorption process.