Detection of activation of the contact system of coagulation in vitro and in vivo: quantitation of activated Hageman factor-C-1-inhibitor and kallikrein-C-1-inhibitor complexes by specific radioimmunoassays.

Radioimmunoassays (RIAs) for the detection of C-1-inhibitor (C-1-Inh) complexed to either kallikrein or activated Hageman factor (factor XIIa) are described. Kallikrein-C-1-Inh or factor XIIa-C-1-Inh complexes were bound to Sepharose to which monospecific antibodies against (pre)kallikrein or factor XII, respectively, were coupled. Bound complexes were subsequently detected by an incubation with affinity purified 125I-labeled antibodies against C-1-Inh. These RIAs were used to detect activation of the contact system of coagulation in vitro and in vivo. Addition of dextran sulfate (DXS) (20 micrograms/ml) to fresh plasma resulted at 37 degrees C in the rapid generation of amidolytic kallikrein activity, which was maximal after 1 to 2 min of incubation and subsequently decreased within a few minutes. The generation of kallikrein activity coincided with the appearance of both kallikrein-C-1-Inh and factor XIIa-C-1-Inh complexes. However, in contrast to kallikrein activity, both types of complexes remained detectable in the incubation mixtures during the incubation period. Experiments with purified kallikrein. C-1-Inh and partly purified beta-factor XIIa, and activation experiments in plasmas deficient in either factor XII or prekallikrein, demonstrated the specificity of both RIAs. The minimal amount of DXS that resulted in the generation of measurable amounts of both types of complexes in plasma was 2-3 micrograms per ml. Similar experiments with kaolin showed that with limiting amounts of activator (1-2 mg/ml), only kallikrein-C-1-Inh complexes were detected in plasma. When larger amounts of kaolin were added to plasma, factor XIIa-C-1-Inh complexes were additionally detected in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of erythrocyte membrane components in hemoglobin-based blood substitutes.

Two methods for the detection of membrane components in human stroma-free hemoglobin solutions are described. The first is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml hemoglobin-solution. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). These methods were used to determine the purity of Hb solutions prepared in two different ways. Hb solutions prepared by filtration of red blood cells, gradually swollen in hypotonic buffer, contained 0.25% of the original amount of phospholipid and no detectable glycophorin alpha. For Hb solutions prepared in a similar way from red blood cells lysed in water, the values for phospholipid and glycophorin alpha were 2.5% and 0.06%, respectively. The determination of both glycophorin alpha and phospholipid gives a useful indication of the purity of Hb solutions.

Function of isolated rabbit hearts perfused with erythrocyte suspension is not stable but improvement may be feasible with hemoglobin solution.

In the present study isolated rabbit hearts were perfused with erythrocyte suspensions (hematocrit 21.5 +/- 0.5%) or hemoglobin solutions according to Langendorff with a constant flow at 37 degrees C. In preliminary experiments three types of stroma-free hemoglobin were used: unmodified, but carefully purified, stroma-free hemoglobin (SFHb), HbNFPLP which is a chemically modified Hb molecule and polyHbNFPLP which is a polymer of HbNFPLP. In hearts perfused with erythrocyte suspensions left ventricular developed pressure and oxygen consumption decreased and perfusion pressure increased steadily from the beginning of the perfusion. Dark spots appeared on the surfaces of these hearts, which were the result of extravasation of erythrocytes. As a consequence capillaries probably became obstructed, leading to reduced cardiac function. Hearts perfused with stroma-free hemoglobin solutions showed an initial increase in left ventricular developed pressure after switching from Tyrode perfusion to perfusion with hemoglobin solutions. Left ventricular developed pressure and perfusion pressure were stable for about 2 hours in hearts perfused with SFHb and were reasonable for 2 hours when the heart was perfused with HbNFPLP or more than 4 hours with polyHbNFPLP. More extensive experiments with stroma-free hemoglobin solutions when these become available in sufficient quantities have, according to the results from preliminary experiments, the potential of showing good oxygen supply resulting in reasonable cardiac function.

Oxygen affinity of hemoglobin solutions modified by coupling with NFPLP and the effects on tissue oxygenation in the isolated perfused rat liver.

From these liver perfusions with Hb and Hb/HbNFPLP solutions the following conclusions can be drawn: In spite of the chemical modification of the hemoglobin molecule, no rheological differences are seen. All parameters measured were sensitive to hypoxia induced by a decrease in perfusion flow rate. The NFPLP-induced decrease in oxygen affinity was reflected in a higher venous PO2. These in-vivo observations are in agreement with the in-vitro measured oxygen dissociation curves. The difference in PO2 did not result in a change in the other oxygen-sensitive parameters in this model under the chosen conditions. Possible causes for these observations are: the level of hypoxia was too low the oxygen supply in the perfusions with the modified hemoglobin solutions was lower than the oxygen supply in the perfusions with normal hemoglobin. Whether or not this observation is due to an intrinsic property of the modified hemoglobin molecule remains to be established.

The influence of the position of the oxygen dissociation curve on oxygen-dependent functions of the isolated perfused rat liver. II. Studies at different levels of hypoxia induced by decrease of blood flow rate.

The influence of the 2,3-diphosphoglycerate (2,3-DPG) induced displacement of the oxygen dissociation curve (O.D.C.) on the isolated perfused rat liver was studied at different levels of stagnant hypoxia (hypoxia induced by decrease of blood flow rate at constant PO2 and at constant haematocrit). Rat livers were perfused with a medium containing either fresh or 2,3-DPG-depleted erythrocytes (2,3-DPG content: 4.3 +/- 0.4 and 0.6 +/- 0.4 mmol/l erythrocytes, respectively). The difference in oxygen affinity of the red cells did not affect the vascular resistance of the perfused liver tissue. The decrease in oxygen supply brought about by a decrease in blood flow rate resulted in a decrease of bile flow rate and of oxygen consumption. The higher 2,3-DPG content of the fresh erythrocytes was reflected in a higher bile flow rate at all blood flow levels, in a higher oxygen consumption at lower blood flow levels, and in a higher venous PO2 at the higher blood flow levels. Venous PO2, lactate/pyruvate (L/P) ratio and beta-hydroxybutyrate/acetoacetate (betaOH/Acac) ratio were relatively insensitive to stagnant hypoxia and to a difference in the 2,3-DPG content of the erythrocytes. The ATP content of the liver tissue was decreased at the lower blood flow levels. However, the ATP content of the livers perfused with fresh erythrocytes did not differ from that of the livers perfused with 2,3-DPG-depleted erythrocytes.

Prolonged vascular retention of a hemoglobin solution modified by cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate.

The usefulness of hemoglobin solutions as plasma expanders with oxygen-carrying capacity is limited by a high oxygen affinity and a rapid clearance from the circulation. A large amount of the hemoglobin is cleared by the kidneys, because the hemoglobin can pass the glomeruli after dissociation into dimers. This dissociation can be prevented by cross-linking the beta chains with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP), a modification that also diminishes the oxygen affinity. In the present study, the vascular retention of modified hemoglobin (HbNFPLP) compared with unmodified hemoglobin (Hb) was investigated in rats and rabbits by replacing half the blood volume with a mixture of Hb and HbNFPLP (7 gm/100 ml). The amount of free hemoglobin in the circulation was determined from the plasma concentration, corrected for the decrease in plasma volume. The decrease in plasma volume was calculated from the increase in hematocrit (in the rats, 30% to 40% in 3 hours). The ratio Hb/HbNFPLP was determined by high-performance anion-exchange chromatography. The half-disappearance times for HbNFPLP and Hb were found to be 3 hours and 1 hour in rats and 7 hours and 2.5 hours in rabbits, respectively. In the rats, one third of the unmodified Hb was found in the urine 5 hours after the exchange, against only 5% of the HbNFPLP. In rats without kidneys, the ratio of Hb/HbNFPLP in the circulation remained constant. The results demonstrate that intramolecular cross-linking of hemoglobin with NFPLP prevents excretion by the kidneys, but does not influence other clearance mechanisms.

Endotoxin in blood products: correlation between the Limulus assay and the rabbit pyrogen test.

The possibility of replacing the rabbit pyrogen test by the Limulus (LAL) test, as a final release test for plasma products, was investigated. The LAL test measured the endotoxin content quantitatively, using a chromogenic substrate. The samples were boiled and centrifuged to remove inhibiting substances, which represent a major problem when testing plasma protein samples. In order to correlate the LAL test to the rabbit test, parallel assays were performed on 85 batches of two different human albumin preparations. For both products, a positive correlation was observed between the two tests. However, the pass/fail limit of the rabbit test was found at different endotoxin levels, corresponding to about 2 and 20 ng/kg, respectively. This discrepancy could be removed by extracting the endotoxin before administration to rabbits. It is concluded that the endotoxin, detected in plasma products by the LAL test, may be present in a non-pyrogenic state.

The influence of the position of the oxygen dissociation curve on oxygen-dependent functions of the isolated perfused rat liver. III. Studies at different levels of anaemic hypoxia.

The influence of a 2,3-diphosphoglycerate (2,3-DPG)-induced displacement of the oxygen dissociation curve (O.D.C.) on the isolated perfused rat liver was studied at different levels of anaemic hypoxia. Rat livers were perfused either with fresh or with 2,3-DPG-depleted human erythrocytes at different haematocrit values (from 30% to 2.5%) at constant Po2 of the inflowing perfusate and at constant blood flow rate. The 2,3-DPG-induced difference in oxygen affinity of the red cells did not cause a significant difference in perfusion pressure during the perfusion experiments. Therefore, there is no evidence that 2,3-DPG did alter the vascular resistance of the liver, since blood flow rate could be adusted at equal values. The decrease in oxygen supply brought about by decrease of haematocrit caused a decrease of O2 consumption, of bile flow rate and of venous Po2 and an increase of lactate/pyruvate (L/P) ratio and of beta-hydroxybutyrate/acetoacetate (betaOH/Acac) ratio. There was no influence of a difference in 2,3-DPG content of the erythrocytes on the above-metioned parameters during severe anaemic hypoxia. At moderate anaemic hypoxia the venous Po2 was higher during perfusion with fresh erythrocytes than during perfusion with 2,3-DPG-depleted erythrocytes. Thus, although 2,3-DPG may play a compensatory role during conditions of mild anaemia, no such effects can be observed during conditions of severe hypoxia.

Key role of macrophages in hypotensive side effects of immunoglobulin preparations. Studies in an animal model.

Intravenous administration of certain immunoglobulin preparations may cause severe adverse reactions, especially in hypogammaglobulinaemic patients. Because the exact mechanism of the adverse reactions is still unknown, we investigated the severe, prolonged hypotension induced in anaesthetized rats on rapid i.v. infusion of standard immunoglobulin preparations. The hypotensive response was previously shown to be associated with IgG aggregates in the preparations but independent of complement activation. We found that the hypotension could be prevented by treating the rats with a specific receptor antagonist of platelet-activating factor; or by depletion of the macrophages of the rats; or by pretreatment with monomeric IgG. This provided evidence that the hypotension is initiated by interaction of IgG-aggregates with Fc-receptors on macrophages, leading to the production of platelet-activating factor. We conclude that the rat model provides a sensitive and reproducible test system for macrophage-activating properties of immunoglobulin preparations for i.v. administration which may lead to vasoactive side effects.

The influence of the position of the oxygen dissociation curve on oxygen-dependent functions of the isolated perfused rat liver. I. Studies at different levels of hypoxic hypoxia.

Isolated rat livers were perfused with fresh and 2,3-DPG (2,3-diphosphoglycerate)-depleted human erythrocytes at different levels of hypoxia. The mean P50 values of the measured actual oxygen dissociation curves (O.D.C.) were 24.5 and 18 mm Hg. No changes in flow rate and perfusion pressure occurred under the different experimental conditons. It was shown that an advantage or disadvantage of a shift of the O.D.C. depends on the degree of hypoxia, as reflected in the venous PO2. Perfusions with fresh erythrocytes showed higher venous PO2 values during normoxia or moderate hypoxia and lower venous PO2 values at severe hypoxia. A cross-over point was found at a PO2 in the portal vein of 36 mm Hg. The disadvantage of perfusions with fresh erythrocytes at severre hypoxia was also reflected in higher cytoplasmatic and mitochondrial redox levels. Using bile flow rate as an indirect measure for the rate of hydroxylation-dependent O2 consumption a favourable effect of perfusion with fresh erythrocytes was found at a PO2 in the portal vein of 100 and 40 mm Hg.

Neonatal pneumothorax drainage systems: in vitro evaluation.

We studied, in vitro, different commercially available components for pneumothorax drainage, i.e. drainage tubes, Heimlich flutter valve and vacuum control units. The drainage of a pneumothorax by a drainage tube was, as expected, directly dependent on Poiseuille's law and was influenced more by diameter than length. Of practical importance, a size 6 French gauge tube, used for the very small newborn, may not efficiently evacuate a pneumothorax due to a large air leak. The Heimlich flutter valve, though useful clinically, adds to the resistance of the system especially if fluids accumulate in the valve. All vacuum control units, adaptations of the basic three- or four-bottle pleural drainage system, functioned adequately but simple changes in construction may increase the safety of some of these systems.

Effect of intraperitoneal administration of two different batches of albumin solutions on peritoneal solute transport in CAPD patients.

The effects of intraperitoneal administration of two different batches of human albumin (batch A and batch B) on peritoneal solute transport and dialysate white cell count were studied in 16 CAPD patients. The studies were done on two separate days during a 4-h dwell, one day without and one day with the intraperitoneal administration of 10 g/l human albumin. Marked differences were found between the two batches. The transport of all measured solutes increased during administration of batch A compared to the control experiments: urea 78% +/- 62%, lactate 51% +/- 38%, creatinine 96% +/- 54%, glucose 67% +/- 55%, inulin 27% +/- 33% IgG 126% +/- 80%, mean +/- SD; P less than 0.02). In the experiments with batch A the white cell count of the test bag was greater than that of the effluent ('night bag') before the test (13 +/- 5 vs 194 +/- 61 mm3/l; P less than 0.02). These effects were also observed when the dialysate was buffered to pH = 7.4 before inflow. Albumin batch B showed no effect on solute transport and white cell count. The effects of solute transport and white cell count are probably caused by the greater concentration of prekallikrein activator in batch A when compared to batch B (30.1 vs 0 U/l). Caution is warranted when human albumin is used for simultaneous measurement of peritoneal fluid and solute kinetics.

Effect of coupling of 2-nor-2-formylpyridoxal 5'-phosphate to stroma-free hemoglobin on oxygen affinity and tissue oxygenation. Studies in the isolated perfused rat liver under conditions of normoxia and stagnant hypoxia.

Hemoglobin in stroma-free solution (7 gm/100 ml) was modified by covalently cross-linking the beta-chains with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP). The coupling efficiency was approximately 65%. The oxygen dissociation curve of the coupling mixture was shifted to the right, with a P50 of 30 mm Hg vs. 15 mm Hg for the nonmodified solution. The effect of the modification on tissue oxygenation was studied in the isolated perfused rat liver at normoxia and at hypoxia induced by decrease of flow rate at constant Po2 and hemoglobin concentration. The two perfusates used were a nonmodified hemoglobin solution and the coupling mixture. The chemical modification of the hemoglobin molecule did not affect the vascular resistance in the liver tissue. During normoxia the NFPLP-induced decrease in oxygen affinity was reflected in a higher venous Po2. The differences in the other oxygen-sensitive parameters were not significant. The decrease in O2 supply induced by a decrease of perfusion flow rate (stagnant hypoxia) resulted in a decrease in venous Po2, O2 consumption, and bile flow rate, and an increase in the cytoplasmatic redox level (lactate/pyruvate ratio) and the mitochondrial redox level (beta-hydroxybutyrate/acetoacetate ratio). During hypoxia the changed oxygen affinity of the modified hemoglobin solution was reflected in small but significant differences between both perfusates in the venous Po2 and both redox levels. No change in O2 consumption and bile flow rate was observed. When compared with earlier low-flow perfusions of the isolated rat liver with erythrocytes, the oxygen affinity of hemoglobin solutions appears not to be rate limiting for the O2 consumption, probably because of better tissue perfusion with hemoglobin solutions.

Biophysical characteristics of hemoglobin intramolecularly cross-linked and polymerized.

Hemoglobin modified by intramolecular cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) has potential application as an oxygen-carrying plasma expander with improved vascular retention time (threefold) and oxygen-transporting properties compared to native hemoglobin. Under some conditions a further prolongation is required. In this study polymerization of the purified modified hemoglobin (HbNFPLP) with glutaraldehyde was investigated. The influence of the degree of polymerization on the iso-oncotic concentration and the viscosity of the polymerized Hb-NFPLP (polyHbNFPLP) was investigated with three products polymerized to an increasing extent. Exchange transfusions in rats followed by gel filtration analyses of plasma samples provided information on the vascular retention time of four separate polymer fractions (i.e., monomers, dimers, trimers/tetramers, and polymers). The vascular retention time of these fractions correlated with their size. For lightly and highly polymerized HbNFPLP we found fivefold and sevenfold increases, respectively, compared to the retention time for native hemoglobin. This finding and the different shapes of the clearance curves suggested a different clearance mechanism from the vascular system for the monomer and polymer fractions. By polymerizing HbNFPLP the oxygen affinity was enhanced (oxygen half-saturation pressure shifted from 45 to 22 mm Hg), and it appeared to be independent of the degree of polymerization. Because hemoglobin was undetectable in the urine of the rats after the exchange transfusions, no accumulation of polyHbNFPLP will occur in the tubules of the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

"Shake hands"; diagnosing a floppy infant--myotonic dystrophy and the congenital subtype: a difficult perinatal diagnosis.

Myotonic dystrophy is a multi-organ disease inherited in a complicated way. Congenital myotonic dystrophy is a distinct entity with severe symptoms leading to a high rate of perinatal morbidity and mortality. The occurrence of congenital myotonic dystrophy often allows a subsequent diagnosis in the mother with important implications for her life, her further pregnancies and offspring. Genetic principles of anticipation and somatic mosaicism are involved and hamper the prenatal diagnostic possibilities. A family is presented in which maternal myotonic dystrophy and congenital myotonic dystrophy were diagnosed after the third pregnancy. The key features leading to the diagnosis were obstetric history, neonatal hypotonia and asphyxia, facial abnormalities in the mother together with the inability to bury eyelashes and delayed release of grip after shaking hands. The disorder is reviewed with respect to clinical symptoms, pathogenesis and genetics.

Bradykinin-mediated hypotension after infusion of plasma-protein fraction.

In patients who required volume expansion during extracorporeal circulation, the plasma bradykinin concentration was monitored simultaneously with the mean arterial pressure during infusion of either albumin solution or PPF. The PKA content of the PPF and the albumin solution was 29 and 3 U/L, respectively, measured spectrophotometrically. In six patients receiving 250 ml of PPF, the mean arterial pressure decreased 22% to 54% within 1.5 min after infusion, whereas the plasma bradykinin concentration, measured by radioimmunoassay, increased significantly (p less than 0.0005) during the first minute. In six patients receiving 250 ml of 4% albumin solution, no blood pressure changes were found, and the plasma bradykinin concentration rose only slightly. In vitro, linear correlation (r = 0.94, p less than 0.0005) was observed between the level of PKA of 26 different lots of PPF and the concentrations of bradykinin that were generated in Hageman factor-deficient plasma after incubation with PPF. It is concluded that the hypotensive reactions observed after PPF infusion during extracorporeal circulation are caused by the PKA-induced bradykinin generation.