Sodium bicarbonate alters protein stability and blood coagulability in a simulated Impella purge gap model.

BACKGROUND: The Impella® microaxial blood pumps utilize purge fluid containing heparin to prevent biofouling of internal surfaces. Purge fluid interfaces with blood or blood components at two notable internal locations: (1) 5-8 µm radial gap ("Radial Gap" or "Gap 1") between the motor shaft and bearing, a site accessible by blood proteins or small molecules; and (2) 100 µm axial gap ("Axial Gap" or "Gap 2") between the impeller rotor and bearing, the site of mixing with larger circulating blood components. Despite its efficacy, heparin in the purge fluid complicates overall patient anticoagulation management. Here, we investigate sodium bicarbonate as an alternative to heparin in the purge fluid in a simulated purge gap micro-environment. METHODS: To assess protein stability simulated at Gap 1, human serum albumin (HSA; 40 mg/ml) species were quantified utilizing size exclusion liquid chromatography (SEC-HPLC) after stirring with purge fluid (5% dextrose in water (D5W) with heparin (25 U/ml) or sodium bicarbonate (25 or 50 mEq/L)) over a 24-h period. pH measurements were taken immediately prior to stirring. Mixing between blood and purge fluid at Gap 2 was mimicked in vitro utilizing a 60:40 blood: purge fluid ratio. Purge fluid consisted of D5W with or without sodium bicarbonate (25 or 50 mEq/L). Human citrated blood samples were freshly collected with or without the addition of heparin (5 U/ml). Coagulability was determined via thromboelastography (TEG). pH measurements of blood mixtures were taken immediately before and after TEG analysis. RESULTS: Sodium bicarbonate alone or synergistically with heparin was effective in increasing protein stability, increasing pH, and reducing coagulability. In the Gap 1 model, sodium bicarbonate led to preservation of HSA monomer after 24 h mixing, with monomer composing 88.3 ± 2.3% and 88.6 ± 0.9% of total HSA species for 25 or 50 mEq/L sodium bicarbonate, respectively. Only 60.4 ± 4.3% monomer was observed with D5W alone (p < 0.005). HSA aggregates and fragments were evident in heparin and D5W purge mixtures, but absent in sodium bicarbonate (25 and 50 mEq/L). pH of HSA mixtures significantly increased in the presence of sodium bicarbonate. In the Gap 2 model, combined heparin (5 U/ml) and sodium bicarbonate prolonged clotting time (TEG-ACT), leading to an average increase of 795 ± 275 s (p = 0.04) and 846 243 s (p = 0.03). This trend of reduced coagulability was similarly observed in clot initiation time (R time), clot formation time (K time), and clotting rate (α angle). Blood mixture pH measurements increased with addition of sodium bicarbonate in both heparinized and non-heparinized blood samples. CONCLUSION: Sodium bicarbonate in the purge fluid has the potential to significantly increase protein stability and reduce protein denaturation at the Impella® radial gap (Gap 1), while reducing blood coagulation at the Impella® axial gap (Gap 2). The influence of sodium bicarbonate on the biochemical environment of the purge fluid may ensure stable purge flow resistance and play a synergistic or supportive role in the purge gap micro-environment when used with systemic anticoagulation.

Rapid selection of single-stranded DNA aptamers binding Staphylococcus epidermidis in platelet concentrates.

Staphylococcus epidermidis is the most common transfusion-associated pathogen contaminating platelet concentrates. Methods to reduce or eliminate contaminating bacteria from platelet units are critical for improving the safety of blood transfusions. We used rapid isolation of DNA aptamers (RIDA) to identify single-stranded (ss)DNA aptamers as ligands that specifically bind to S. epidermidis. Five target-specific ssDNA aptamers (76 mer) were obtained under stringent selection conditions. Aptamer SE43 demonstrated higher binding affinity compared with scrambled control. Furthermore, when binding assays were conducted in platelet concentrate, there was a twofold increase in binding affinity compared with the SE43 binding in buffer alone. Our data identified an aptamer that may be useful as a ligand to capture, detect or remove S. epidermidis contaminant from platelet concentrates.

Visualization of exogenous delivery of nanoformulated butyrylcholinesterase to the central nervous system.

Butyrylcholinesterase (BChE) is an efficient bioscavenger of highly toxic organophosphorus poisons and nerve agents. However, BChE administered into the periphery does not provide significant protection of the central nervous system (CNS) due to rejection by the blood-brain barrier. In this study, we evaluated the feasibility of delivering BChE to the CNS by packing it into a block ionomer complex of nanoscale size with a cationic poly(l-lysine)-graft-poly(ethylene oxide) (PLL-g-PEO) copolymer. The multimolecular structure of BChE/PLL-g-PEO complexes was further reinforced by formation of cross-links between the polymer chains. The resulting cross-linked complexes were stable against dilution without significant loss of BChE enzymatic activity. In some cases the BChE was labeled with fluorescent IRDye 800CW before it was incorporated into nanoparticles. BChE/PLL-g-PEO complexes were injected into mice intramuscularly and intravenously. In vivo imaging showed incorporation of the fluorescently labeled BChE in brain. Activity assays showed that BChE remained active in the brain at 72-h post-injection. It was concluded that nanocomplexes can deliver the 340 kDa BChE tetramer to the brain.

Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice.

Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 microm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 microm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

A low-cost interface for multi-electrode array data acquisition systems.

Multi-electrode array systems have enabled the in vitro electrophysiological study of neuronal networks. The data processing component of these systems consists of an advanced computer system and data acquisition electronics that collectively cost more than the multi-electrode arrays and amplifiers. Considering that these elaborate systems may be cost-prohibitive for many laboratories, we have developed a simple but novel method for recording groups of related multi-electrode array channels with a low-cost data acquisition system.