Benjamin Chiang

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.

Initial experience with the AbioCor implantable replacement heart at the University of Louisville.

Potential benefits of heart transplantation are limited by the severe donor organ shortage. The AbioCor implantable replacement heart has been developed as a potential alternative to heart transplantation. We report our initial experience with the AbioCor in a bovine model. A right thoracotomy was performed for access to the heart and great vessels. After initiation of cardiopulmonary bypass, excision of the native ventricles was followed by orthotopic placement of the IRH and complete implantation of the transcutaneous energy transfer coil, controller, and battery pack. Invasive monitoring of IVC, SVC, carotid artery, pulmonary artery, and left atrial (LA) pressures was performed in all animals. Twelve calves have undergone implantation of the AbioCor. There were three early deaths, one from bleeding, one from respiratory failure, and one from neurodysfunction from low flow during CPB. Nine animals have had a normal recovery and survived a mean of 24.5 days (range, 4–48 days). All the animals have demonstrated excellent hemodynamics with the maintenance of normal pressures in the LA, SVC, IVC, pulmonary artery, and aorta. Adjustment of the right-sided internal hydraulic fluid shunt has allowed for control of right-left balance and, thereby, manipulation of left and right side filling pressures. Late morbidity has consisted of neck wound infection and sepsis, pneumonia, and bleeding. Successful orthotopic implantation of all components of the AbioCor has been achieved in a bovine model. This device has demonstrated restoration of normal hemodynamics and excellent function of the atrial hydraulic shunt to achieve right-left balance.

The uptake and metabolism of fructose‐1,6‐diphosphate in rat cardiomyocytes

Fructose‐1,6‐diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N2/5% CO2), each time period followed by a 1 h reoxygenation (95% air/5% CO2). The uptake of FDP by rat cardiomyocytes was more concentration‐dependent than time‐dependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5–10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.

Using fructose-1,6-diphosphate during hypothermic rabbit-heart preservation: a high-energy phosphate study.

BACKGROUND In this study, we evaluated the effects of fructose-1,6-diphosphate (FDP) on high-energy phosphate metabolism during 18-hour hypothermic rabbit-heart preservation. METHODS Under general anesthesia and artificial ventilation, hearts from 42 adult New Zealand white rabbits were harvested, flushed, and preserved in St. Thomas solution at 4(o)C for 18 hours. In the study group (n = 15), FDP (5 mmol/liter) was added to the St. Thomas solution, whereas in the control group (n = 17), fructose (5 mmol/liter) was added. Another 10 hearts did not undergo hypothermic storage, but were used as the normal group for high-energy phosphate concentration comparison. RESULTS After 18 hours of hypothermic preservation, myocardial high-energy phosphate content decreased in both preservation groups. In the study group, left ventricular adenosine triphosphate (ATP) content was 33% of that in the normal hearts, but in the control group, ATP decreased to 14% of normal. Adenosine diphosphate (ADP) content, energy charge, and ATP-to-ADP ratio showed similar decreases. The high-energy phosphate profile (content in the atria and ventricles and the ratio of ATP to ADP to AMP) was maintained in the study group but not in the control group. High-energy phosphate metabolites such as inosine monophosphate (IMP), inosine, and hypoxanthine increased in both preservation groups, but the increase was more prominent in the control group. CONCLUSION Adding FDP to St. Thomas solution attenuated the depletion of high-energy phosphate concentration in the preserved hearts. This difference was especially prominent in the left and right ventricles. The protective effect of FDP during hypothermic heart preservation deserves further study.

Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers

Fructose-1,6-bisphosphate (FBP) is a high-energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects of FBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with 14C-FBP at 30°C. Uptake of FBP was significant (P<0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane, bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P<0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P<0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane-permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl-1,3,5-hexatriene (DPH) or trimethylammmonia-DPH (TMA-DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P<0.05), had no effect on FA of TMA-DPH in the liquid crystalline state of DPPC, but increased the FA of TMA-DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA-DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P<0.05). The addition of 50 mM FBP caused a significant decrease (P<0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head-group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3′-dipropylthiacarbocyanine iodide (DiSC3) and then added FBP. FBP caused a significant, dose-dependent decrease in DiSC3 fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.