P001 - Sepsis impairs the capillary response within hypoxic capillaries and decreases erythrocyte oxygen-dependent ATP efflux

Unfortunately, the original version of this supplement [1] contained errors in two of the abstracts; P037 and P127. Please see details below.

The release of ATP from red blood cells (RBC) in response to low O2 levels is linked to ATP production and the oxygenation state of hemoglobin. Because O2 is unloaded from the RBC, the concentration of deoxygenated hemoglobin increases, displacing phosphofructokinase from the cytoplasmic domain of band 3. We hypothesize that the ATP molecules produced through this glycolytic stimulation at the membrane surface result in the release of ATP from the RBC. Rat whole blood exposed to 5 min of low PO2 in vitro increased plasma [ATP] by 1.0 miccroM (+45%). This increase was reduced to 0.1 microM (+12%, P < 0.05) after citrate incubation and reversed after fluoride treatment (both glycolytic inhibitors) by -0.2 microM (-23%, P < 0.05). Plasma [ATP] of control RBC decreased -0.3 microM (-12%) when 8% CO (P < 0.05) was added to the chamber. Because CO and O2 bind competitively to heme, these results support our hypothesis that the release of ATP from RBC is linked to ATP production through the oxygenation state of the hemoglobin molecule.

Erythrocyte deformability has been recognized as a determinant of microvascular perfusion. Because nitric oxide (NO) is implicated in the modulation of red blood cell (RBC) deformability and NO levels increase during sepsis, we tested the hypothesis that a NO-mediated decrease in RBC deformability contributes to decreased functional capillary density (CD) in remote organs. With the use of a peritonitis model of sepsis in the rat [cecal ligation and perforation (CLP)] and aminoguanidine (AG) to prevent increases in NO, we measured CD in skeletal muscle (intravital microscopy), mean erythrocyte membrane deformability (; micropipette aspiration), systemic NO production [plasma nitrite/nitrate (NO(x)) chemiluminescence], and NO accumulation in RBC [NO bound to hemoglobin (HbNO) detected by electron paramagnetic resonance spectroscopy]. In untreated CLP animals relative to sham, NO(x) increased 254% (P < 0.05), stopped flow capillaries increased 149% (P < 0.05), and decreased 12.7% (P < 0.05), with a subpopulation (5%) of RBC with deformabilities below the normal range. AG prevented increases in NO(x), accumulation of HbNO, and decreases in both and functional CD. We found no evidence of leukocyte plugging postcapillary venules. Our findings suggest that decreased functional CD during sepsis resulted from a NO-mediated decrease in erythrocyte deformability.

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.

Under storage conditions, red blood cells (RBCs) undergo significant biochemical and biomechanical changes that compromise their ability to unload oxygen to the tissues. The clinical benefit of RBC transfusions are, therefore, less than anticipated and may lead to tissue injury rather than improvement. The development of an animal model is an important step in characterizing the loss of effective oxygen availability and assessing the efficacy of transfusion therapy. The aim of this study was to determine when stored rat RBCs develop a storage-lesion similar to human RBCs near the end of their shelf life.

`Rigid' red cells in sepsis are thought to play a role in multiorgan failure by plugging the microvasculature and compromising oxygen delivery. During sepsis endogenous nitric oxide (NO) production is increased. What effect this has on erythrocyte deformability (RBCd) is unclear. We report the effects on RBCd and capillary blood flow when NO overproduction was prevented in septic rats.