Effect of active oxygen radicals on protein and carbohydrate moieties of recombinant human erythropoietin.

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radicals generated from a Fenton system in the presence of 0.016 mM H2O2, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H2O2). The tryptophan destruction was detected at 0.016 mM H2O2 and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increased of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.

Layer-by-layer modification of cation exchange membranes controls ion selectivity and water splitting.

The present study investigates the possibility of inducing monovalent ion permselectivity on standard cation exchange membranes, by the layer-by-layer (LbL) assembly of poly(ethyleneimine) (PEI)/poly(styrenesulfonate) (PSS) polyelectrolyte multilayers. Coating of the (PEI/PSS)N LbL multilayers on the CMX membrane caused only moderate variation of the ohmic resistance of the membrane systems. Nonetheless, the polyelectrolyte multilayers had a substantial influence on the monovalent ion permselectivity of the membranes. Permselectivity comparable to that of a commercial monovalent-ion-permselective membrane was obtained with only six bilayers of polyelectrolytes, yet with significantly lower energy consumption per mole of Na(+) ions transported through the membranes. The monovalent ion permselectivity stems from an increased Donnan exclusion for divalent ions and hydrophobization of the surface of the membranes concomitant to their modification. Double-layer capacitance obtained from impedance measurements shows a qualitative indication of the divalent ion repulsion of the membranes. At overlimiting current densities, water dissociation occurred at membranes with PEI-terminated layers and increased with the number of layers, while it was nearly absent for the PSS-terminated layers. Hence, LbL layers allow switching on and turning off water splitting at the surface of ion exchange membranes.

Catalytic polyelectrolyte multilayers at the bipolar membrane interface.

Bipolar membranes are laminated anion and cation exchange membranes that split water at their interface very efficiently upon application of an electric field. This paper investigates the layer-by-layer (LbL) deposition of polyelectrolyte multilayers, as a tool to introduce molecularly thin catalyst groups at this interface of bipolar membranes. The bipolar membranes were prepared by first modifying an anion exchange membrane by consecutive dipping LbL assembly, then casting a thin highly charged intermediate layer followed by casting a cation exchange layer. The results reveal that polyelectrolytes of higher charge density coated on the anion exchange layer yield better performance. Several parameters of the LbL interface deposition were varied. Out of the investigated LbL assembly parameters, ionic strength and number of layers have shown the largest influence on catalytic activity as well as ionic selectivity. The membrane with two bilayers of poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) and poly(ethyleneimine) (PEI), where the PEI was prepared in 0.5 M NaCl, gave rise to the best performance. Surprisingly, detailed data analysis at low electrical potential suggests that the interface layers of a bipolar membrane play a major role in its permselectivity. Previously, only the bulk thickness of the anion and cation exchange membrane was assumed to influence the bipolar membrane selectivity.

Systemic thromboembolic complications after laparoscopic splenectomy for idiopathic thrombocytopenic purpura in comparison to open surgery in the absence of anticoagulant prophylaxis.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) in adults has a chronic course and may necessitate splenectomy. The current study was undertaken to study the systemic thromboembolic complications of laparoscopic splenectomy (LS) versus open splenectomy (OS) in patients with ITP at two large referral hospitals. PATIENTS AND METHODS: We conducted a retrospective analysis of 49 patients who underwent splenectomy (21 LS and 28 OS) for primary/relapsing refractory ITP between June 1995 and November 2004. Clinically and/or radiologically confirmed deep venous thrombosis (DVT) and/or pulmonary embolism (PE) were assessed within 2 weeks before and after splenectomy. None had prophylactic anticoagulants immediately after surgery. Follow up of those who developed complications continued for at least 2 additional years to assess for contributing factors that may have been masked at the time of occurrence. RESULTS: Two (9.5%) LS group had acute PE within 5 days of LS and their platelet count reached 500A103/I(1/4)L within 4 days and 1000A103/I(1/4)L within 7 days after surgery. Three conversions to OS occurred; none had VTE. DVT occurred in 3 patients (10.7%) in the OS group; none were life threatening. There were no deaths. CONCLUSION: Life-threatening venous thromboembolic events are serious complications after LS and OS for ITP patients if prophylactic anticoagulants are not administered. Patients at risk are those who both have an exponential rise of the platelet count, although factors other than the platelet count may be contributing in OS. Postsplenectomy, ITP should be considered as a thrombophilic condition and studies of additional measures to prevent such events are warranted.