Shaul Yedgar

Molecular weight, shape and structure of mixed micelles of Triton X-100 and sphingomyelin

Abstract The sedimentation coefficients of mixed micelles of Triton X-100 and sphingomyelin have been measured for Triton molar fractions between 0.32 and 0.79. These measurements, together with previously reported diffusion coefficients, are used to calculate the molecular weights of the mixed micelles, and their aggregation numbers. It is found that the molecular weight decreases steadily with increasing Triton molar fraction. However, the Triton aggregation number remains nearly constant at about 196, so that the decreasing molecular weight is reflected in the sphingomyelin aggregation number, which decreases from 442 to 50 as the Triton molar fraction increases from 0.32–0.79. Intrinsic viscosities of the mixed micelle solutions are found to be independent of Triton molar fractions, with a value of 5.5 ml/g. From these measurements a number of inferences are drawn regarding the micellar shape and structure.

Diffusion coefficients of mixed micelles of Triton X-100 and sphingomyelin and of sonicated sphingomyelin liposomes, measured by autocorrelation spectroscopy of Rayleigh scattered light.

Abstract Autocorrelation spectroscopy of Rayleigh scattered light has been used to measure the diffusion coefficients of mixed micelles of Triton X-100 with sphingomyelin. Sphingomyelin is a water-insoluble lipid. It forms a solution of mixed micelles only for molar fractions of Triton X-100 above 0.32. Over the range of Triton X-100 molar fractions from 0.32–1.0 the diffusion coefficient increases from 2.6·10 −7 –4.5·10 −7 cm 2 /s, indicating that the increased concentration ratio of the detergent systematically reduces the size of the mixed micelles. Diffusion coefficients of sonicated liposomes of sphingomyelin were also measured. For specimens of unhydrogenated and partially hydrogenated sphingomyelin, respective diffusion coefficients of 8.8·10 −8 and 3.1·10 −8 cm 2 /s were obtained. The corresponding Stokes radii for the two specimens are 250 and 700 A, respectively.

Thrombolytic therapy reduces red blood cell aggregation in plasma without affecting intrinsic aggregability

Red blood cell (RBC) aggregation may contribute to occlusion of the coronary microcirculation during myocardial infarction. We studied the effect of thrombolytic therapy on RBC aggregation in patients with acute myocardial infarction (AMI). Compared with patients with myocardial infarction who did not receive thrombolytic therapy, those treated with systemic thrombolysis exhibited significantly reduced RBC aggregation, reduced plasma fibrinogen levels and increased plasma D-dimer levels. Using measurement of RBC aggregation in a standardized dextran-500 solution, reduction in RBC aggregation after thrombolysis was shown to be plasma dependent. Thrombolytic therapy had no direct effect on intrinsic RBC aggregability in patients with AMI. We conclude that thrombolytic therapy has rheologic consequences that may contribute to its overall efficacy. Inhibition of RBC aggregation by thrombolytic therapy may result from the degradation of fibrinogen, a key factor in the formation of RBC aggregates, and from the generation of fibrinogen degradation products capable of disaggregating RBCs.

Solubilization of sphingomyelin by triton X-100 effect of the method of mixing and the concentrations of detergent and lipid☆

Abstract Mixed dispersions of sphingomyelin and Triton X-100 were prepared by two procedures. In method A, aqueous dispersions of sphingomyelin were mixed with aqueous solutions of Triton X-100. In method B, solutions of sphingomyelin and Triton X-100 in organic solvent were mixed, the solvent was evaporated and the dry residue was dispersed in buffer. Measurement of turbidities, electron microscopy and sedimentation of the mixed dispersions suggested the following: Below the critical micellar concentration of Triton X-100, the sphingomyelin is present as liposomes which sediment in the ultracentrifuge. Above the CMC, mixed micelles of sphingomyelin and Triton form. Method B resulted in aggregates of sphingomyelin which contain Triton X-100 even below its critical micellar concentration and which are smaller than those obtained by method A.

Resuscitation With Aged Blood Exacerbates Liver Injury in a Hemorrhagic Rat Model

Objective:Blood loss and transfusion are frequent among patients undergoing liver surgery. Concerns have been raised about the safety and efficacy of transfusing stored blood. The influence of transfusing fresh vs. stored blood on the liver has not been studied to date. We tested the hypothesis that transfusion of stored, but not fresh blood, adversely affects liver outcome in vivo following acute hemorrhage. Additionally, possible mechanisms linking adverse liver outcome with increased storage duration were evaluated. Design:Prospective, controlled, animal study. Setting:University research laboratory. Subjects:Adult male Sprague-Dawley rats Interventions:Anesthetized rats were randomized to control, hemorrhagic and shock group (acute bleeding; HSG), or hemorrhagic and blood resuscitation groups (BR) (with fresh blood [BR-d0], blood stored for 4 [BR-d4] or 7 [BR-d7] days, or packed RBCs stored for 7 days [packed RBC-d7]). Measurements and Main Results:Administration of blood or packed RBC stored for 7 days exacerbated liver injury as reflected by liver necrosis and enhanced apoptosis (p < 0.001). Functional MRI analysis of the liver demonstrated significant improvement in liver perfusion with fresh blood (% change in functional MRI signal intensity due to hyperoxia was 16% ± 3% in BR-d0 vs. 4% ± 3% in hemorrhagic group, p < 0.001) but not with stored blood (12% ± 2% and 9% ± 5% for BR-d4 and BR-d7, respectively). Analysis of stored blood showed reduction in RBC deformability at 7 days of storage, reflecting a five-fold increase in the number of undeformable cells. Conclusion:Liver injury is exacerbated by the transfusion of stored blood, primarily due to the change in the rheological properties of RBC. This data call for clinical studies in patients undergoing liver resection or transplantation.

Abatement of bleomycin-induced pulmonary injury by cell-impermeable inhibitor of phospholipase A2

The mechanism of bleomycin (Bleo)-induced pulmonary injury is not fully understood. Elevated levels of lung phospholipase A2 (PLA2) have been previously reported following intratracheal (IT) instillation of Bleo, but the role of this enzyme in the pathogenesis of lung injury is not clear. In this pilot study, we have evaluated the effect of a cell impermeable inhibitor of PLA2 (CME) on Bleo-induced pulmonary inflammation in hamsters. Pulmonary injury was induced by a single IT instillation of Bleo (1 unit/0.5 ml saline). Three groups of male Syrian hamsters were evaluated: 1) BLEO-CME animals received IT Bleo and daily intraperitoneal (IP) injections of CME (1 mumole/kg), starting 1 day before IT instillation; 2) BLEO-SAL animals--received IT Bleo and IP injections of saline and 3) SAL-SAL animals--treated with IT and IP administrations of saline. Animals were sacrificed 14 days after IT treatment and lung injury was evaluated histologically by a semiquantitative morphologic index and by a differential cell count of bronchoalveolar lavage fluid. CME treatment significantly ameliorated Bleo-induced lung injury compared to BLEO-SAL animals (P < 0.05). The percentage of neutrophiles in bronchoalveolar lavage fluid was reduced from 17.7 +/- 3.2% (mean +/- S.E.) in BLEO-SAL group to 7.3 +/- 1.7% in BLEO-CME group (P < 0.05), achieving levels comparable to SAL-SAL control animals. These results suggest that treatment with an extracellular PLA2 inhibitor-CME abates Bleo-induced pulmonary injury. This may indicate an active role of PLA2 in the pathogenesis of interstitial pulmonary fibrosis.

Diabetic foot disease is associated with reduced erythrocyte deformability.

The pathogenesis of diabetic foot disease is multifactorial and encompasses microvascular and macrovascular pathologies. Abnormal blood rheology may also play a part in its development. Using a cell flow analyser (CFA), we examined the association between erythrocyte deformability and diabetic foot disease. Erythrocytes from diabetic patients with no known microvascular complications (n = 11) and patients suffering from a diabetic foot ulcer (n = 11) were isolated and their average elongation ratio (ER) as well as the ER distribution curve were measured.

Increased red blood cell aggregation in patients with Gaucher disease is non-inflammatory.

Red blood cell (RBC) aggregation is enhanced in the presence of ongoing inflammation, because of plasma protein effects, especially fibrinogen. Large RBC aggregates, in addition to being a marker of systemic inflammation, may hinder tissue perfusion and oxygenation. Gaucher disease, the most common lysosomal storage disorder, evinces many of the hallmarks of chronic inflammation. Manifestations of Gaucher disease which may be related to microvascular occlusion include avascular necrosis (AVN), bone crisis, and pulmonary hypertension. This study aims to determine whether increased RBC aggregation in non-splenectomized patients with Gaucher disease is due to Gaucher-related inflammation. The Cell Flow Properties Analyzer (CFA) monitors blood under conditions of different shear stress by creating varying pressure gradients. Blood from non-splenectomized patients with Gaucher disease showed only a slight correlation between aggregation parameters and fibrinogen levels, whereas blood from non-splenectomized patients treated with enzyme replacement therapy (ERT) showed marked correlation between aggregation parameters and fibrinogen, as in the control group. These results underscore the hypothesis that RBC aggregation in Gaucher disease is increased by (at least) two mechanisms: a fibrinogen-mediated inflammatory process and another non-inflammatory process that may be induced by elevated glucocerebroside levels in the RBC and/or inhibited by elevated plasma cerebroside levels.

Plasma dependent reduction in red blood cell aggregation after dextran sulfate low-density lipoprotein apheresis--implications for rheological studies.

Abstract:  Red blood cell (RBC) aggregation is increased in familial hypercholesterolemia, and is reduced significantly after low density lipoprotein (LDL) apheresis. The purpose of the present study was to clarify whether this reduction depends on changes in plasma composition, RBC membrane properties, or both. RBC aggregation was determined in a computerized cell flow‐properties analyzer, before and after LDL apheresis. We compared RBC aggregation in autologous plasma with aggregation in a plasma‐free standard solution (0.5% of dextran 500 kDa) to define the separate contributions of plasma and cellular properties to the observed RBC aggregation. RBC aggregation in autologous plasma was reduced by 35.5% after LDL apheresis (P = 0.01) but was not significantly affected when measured in dextran 500. This suggests that LDL apheresis attenuated RBC aggregation by altering plasma composition rather than RBC membrane properties. These results are relevant to the understanding of hemorheological changes which follow therapeutic apheresis in hypercholesterolemic patients.