Evaluation of a bench-top nephelometric immunoassay analyzer.

We evaluated the Technicon DPA-1 immunoassay analyzer on its analytical characteristics. Therefore we studied three assays: albumin in cerebrospinal fluid and IgG and transferrin in serum. When tested with the Cusum test for linearity albumin, IgG, and transferrin measurements showed no deviation from linearity. Closer examination revealed an abrupt difference of recovery (from 99 to 87%) in the albumin assay when the automatic dilution changed over from the primary analytical to the high analytical concentration range. One calibration was found sufficient for at least 14 days of measurement. Imprecision was well below the critical limits for reproducibility. We found reasonable agreement between the results from the DPA-1 and the results from comparison methods. However, the correlation plot of IgG showed lack of fit at a distinct segment of the regression line. This appeared to be caused by the poor recovery of the DPA-1 at the lower limit of the high analytical concentration range. The assays of IgG and transferrin were found insensitive for interference by hemoglobin, triglycerides, urea, and bilirubin. The albumin assay was found sensitive for bilirubin and triglycerides. No reagent- and sample-to-sample carry-over could be detected in the assays evaluated.

Increased frequency of chromosomal damage in peripheral blood lymphocytes up to nine years following curative chemotherapy of patients with testicular carcinoma.

The presence of micronuclei (MN) in binucleated peripheral blood lymphocytes of 37 testicular carcinoma patients was studied 1.5 to 9.3 years following curative chemotherapy. All patients received cisplatinum, 35 patients also received bleomycin. In addition, most patients were treated with another cytotoxic drug, i.e., vinblastine (n = 23) and/or etoposide (n = 24). The mean time interval between cessation of chemotherapy and the micronucleus assay was 4.6 years. The median frequency of MN in binucleated cells in treated patients (0.059) was significantly higher than that in 12 untreated cancer patients (0.036; P = 0.003) or that in 26 healthy age-matched controls (0.034; P less than 0.001). Frequencies of MN in the 12 untreated cancer patients (including 7 patients with disseminated testicular carcinoma) did not differ from those in the 26 healthy controls (P = 0.890), suggesting that chromosomal damage in lymphocytes of treated testicular cancer patients must be attributed to the chemotherapy. Results indicate that lymphocytes containing chemically induced chromosomal damage persist for up to 9.3 years following cessation of chemotherapy. The implications of these findings with regard to the increased risk for secondary tumors are discussed.

How to study the aetiology of burn injury: the epidemiological approach.

Effective prevention of burn injury should be based on sound aetiological knowledge. This article deals with epidemiological methods to study the incidence of burn injury as a function of its risk factors. Central methodological issues are comparability of baseline prognosis, comparability of measurements (of effects in cohort studies and of risk factors in case-control studies), and comparability of external circumstances. These principles are clarified with a number of fictitious examples of risk factors for burn injury. It is explained that in preventive trials comparability may be achieved by randomization, blinding and placebo intervention. The main tools in non-experimental studies are deliberate selection and multivariate analysis. Special attention is given to the definition of the source population and to reducing measurement incomparability in case-control studies. Some well-designed case-control studies following these principles might bring effective prevention of burn injury some steps nearer.

Correction of instrument- and reagent-based differences in determination of the International Normalized Ratio (INR) for monitoring anticoagulant therapy.

As recommended by the World Health Organization, standardization of prothrombin time assays involves conversion of prothrombin times into International Normalized Ratios (INR). We investigated the effect of two different methods (Nycomed's Thrombotest, and Instrumentation Laboratory's PT-fibrinogen) and three coagulation instruments (Schnitger & Gross, KC-10, and ACL) on calculations of INR. The INR plots showed considerable scatter of individual values around the regression lines when the two different methods were compared. Systematic differences in the outcome of INR calculation were related to the use of the different coagulation instruments. Prothrombin times obtained with the different instruments were linearly correlated. We used the bias of these lines to correct results for both the patients' samples and the reference samples. This correction yielded INR values from the different instruments that agreed well.

Heparin degradation in the endothelial cells.

We analyzed the molecular weight distribution of (125I) heparin fractions bound or internalized by human endothelial cells, using gel permeation chromatography. Our results showed that high molecular weight heparin chains are selectively bound and internalized by endothelial cells. Endocytosis is followed by depolymerization of internalized heparin. Chloroquine prevented depolymerization of internalized heparin, indicating that lysosomal enzymes are involved in this process. Degradation of high molecular weight heparin chains by endothelial cells may contribute to the inactivation of the drug, especially as concerns the antifactor IIa activity.

Selective binding of heparins to human endothelial cells. Implications for pharmacokinetics.

Cultured endothelial cells isolated from human umbilical vein bind heparin and heparin fragments. The binding capacity of endothelial cells for 35S-heparin was for 38% composed of high affinity binding sites (Kd = 11 X 10(-8) M) and for 62% of sites with much lower affinity (Kd = 14 X 10(-7) M). The affinity of unlabeled compounds for heparin binding sites was determined by competition with binding of 125I-heparin. I50 found for unlabeled heparin was 16 X 10(-8) M, which is in agreement with the Kd for binding of heparin to high affinity sites. PK 10169, a low molecular weight fragment of heparin, competed only at relatively high concentrations (I50 = 10(-5) M). Competition experiments with subfractions of PK 10169 showed that I50 was inversely correlated with molecular weight. Gelfiltration of 35S-heparin and 35S-PK 10169 before and after binding to endothelial cells demonstrated a selective binding of high molecular weight molecules from polydisperse heparin and PK 10169 preparations. Bound heparin and PK 10169 molecules were detached from the cell-surface by proteolytic treatment and tested for antifactor-Xa and antifactor-IIa activity. Released heparin is slightly more active in antifactor-Xa and antifactor-IIa activity than its parent preparation. Released PK 10169 was 4 fold more active in antifactor-Xa and 8 fold more active in antifactor-IIa assays than heparin.

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

Role of accessory components in the activation of vitamin K-dependent coagulation factors.

Kinetic studies of prothrombin activation and intrinsic factor X activation carried out in the absence and presence of phospholipids and the protein cofactors Va or VIIIa have provided insight in the mechanism by which the accessory components enhance coagulation factor activation. In intrinsic factor X and prothrombin activation, phospholipids cause a drastic drop of Km for the substrates factor X and prothrombin, whereas the protein cofactors factor Va and factor VIIIa increase Vmax of the prothrombin- and factor X-activating reactions. The mode of action of factor Va in prothrombin activation is however somewhat more complex. Besides its stimulatory effect on the catalytic activity of factor Xa, Factor Va also plays an important role in the assembly of the prothrombin-activating complex at phospholipid surfaces especially when the latter have a low affinity for vitamin-K-dependent coagulation factors. This effect is likely accomplished by promoting the binding of both prothrombin and factor Xa to the procoagulant surface.

Effects of pre-treatment with sodium butyrate on the frequencies of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes.

The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal aberrations were studied in human peripheral blood lymphocytes. Unstimulated (G0) lymphocytes were pre-treated with sodium butyrate (5 mM) for 24 h, X-irradiated and then stimulated to pass through the cell cycle. Cells in their first post-radiation metaphase were scored for chromosomal aberrations. In parallel biochemical experiments nucleoid sedimentation technique was used to examine the induction and repair of DNA-strand breaks. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. We therefore suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G0 lymphocytes.

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus.

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.

Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid.

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa. Our data indicate that the enzymatic unit in the intrinsic factor X activator is a 1:1 stoichiometrical complex of factor IXa and factor VIIIa bound to phospholipid. Titrations with factor IXa at fixed concentrations of phospholipid and factor X show that the apparent dissociation constant of factor IXa for phospholipid is lowered from 10(-6) M to 10(-8) M by the presence of factor VIIIa. We conclude, that in analogy with the role of factor Va in prothrombin activation, phospholipid-bound factor VIIIa functions as a high-affinity binding site ("receptor") for factor IXa in the intrinsic factor X activating complex. Therefore, factor VIIIa increases the observed Vmax of factor X activation by 1) enhancing the kcat of the reaction and 2) increasing the amount of phospholipid-bound factor IXa that participates in factor X activation.

The role of activated human platelets in prothrombin and factor X activation.

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex.

The kinetic parameters of bovine prothrombin activation by factor Xa were determined in the absence and presence of factor Va as a function of the phospholipid concentration and composition. In the absence of factor Va, the Km for prothrombin increases proportionally with the phospholipid concentration and correlates well with the affinity of prothrombin for the different membranes. Phospholipid vesicles with a high affinity for prothrombin yield low Km values compared to membranes with less favorable binding parameters. At limited phospholipid concentrations, the Vmax of prothrombin activation correlates with the binding affinity of factor Xa for the various phospholipid vesicles. Membranes with a high affinity for factor Xa have high Vmax values, while for membranes with a low affinity a low Vmax is observed. Extrapolation of double-reciprocal plots of 1/Vmax vs. 1/[phospholipid] to infinite phospholipid concentrations, a condition at which all factor Xa would participate in prothrombin activation, yields a kcat of 2-4 min-1 independent of the type and amount of acidic phospholipid present in the vesicles. Also, in the presence of factor Va the Km for prothrombin varies proportionally with the phospholipid concentration. There is, however, no correlation between the binding parameters and the Km. Factor Va drastically lowers the Km for prothrombin for vesicles that have a low affinity for prothrombin. Vesicles composed of 20 mol % phosphatidylglycerol and 80 mol % phosphatidylcholine have a Km of 0.04 microM when factor Va is present, compared to 2.2 microM determined in the absence of factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet membrane involvement in blood coagulation.

Intact platelets do not show procoagulant phospholipids on their exterior. These phospholipids are located at the inside leaf of the bilayer membrane. They become available by (a) disrupture of the platelets (mechanical, osmotical etc.), (b) by a mechanism specific for the platelets, that we call the membrane flip-flop. Membrane flip-flop translocates procoagulant phospholipids (mainly phosphatidylserine) in the intact platelet from the inside to the outside. Thus the intact platelet becomes procoagulant. The trigger for the flip-flop mechanism is the simultaneous presence of small amounts of collagen and thrombin. The clotting factors IXa and VIIIa bind to the procoagulant lipids to form the factor X converting enzyme and factor Xa and factor Va to form prothrombinase.

The effect of pH and ionic strength on the steady-state activity of isolated cytochrome C oxidase.

1. The turnover number and apparent Km of isolated beef-heart cytochrome c oxidase were found to increase continuously when the pH was lowered from 8.6 to 4.6 (turnover number 32-630 s-1). In this pH range neither irreversible denaturation of the enzyme nor an optimum for the turnover number was observed. 2. The turnover number of cytochrome c oxidase was found to be independent of ionic strength. It was concluded that the dependence of the activity of cytochrome c oxidase on ionic strength is caused by a change in the value of Km for cytochrome c. 3. The pH dependence of the turnover number of cytochrome c oxidase can be described by a simple model in which at least three sites on the complex of cytochrome c oxidase with cytochrome c (pKa 8.0, 6.5 and 4.8) can take up a proton.