Reduced Fatty Acid Binding Capacity of Human Albumin Used in Volume Resuscitation May Potentially Increase Hemolysis in Hypovolemic Shock

Abstract

The choice of resuscitation fluid for hypovolemic trauma patients in the absence of fresh whole blood remains controversial. Albumin solutions have shown promise in animal studies but meta‐analysis suggests they have had little to no benefit in human trials. This contradiction is currently unresolved. Unbound (cytotoxic) free fatty acids (FFAs) are generated in the intestine during ischemia and enter the mesenteric lymph, from where they likely enter the circulation, resulting in organ damage, hemolysis, coagulopathy and further ischemia. We hypothesize that resuscitation fluids with higher available binding capacities for FFAs may help prevent these events in shock. The objectives of this in‐vitro study were to determine: 1) if commercial sources of pharmaceutical‐grade human albumin contain FFAs, 2) if these FFAs result in a decreased available fatty acid binding capacity, and 3) if binding capacity will affect the level of hemolysis that occurs in whole blood when exogenous FFA is introduced in levels similar to those in shock. To achieve these objectives, we first measured FFA concentrations in human albumin via a kit. We then developed a new assay for measuring fatty acid binding capacity using exogenous oleic acid (OA), glass fiber filtration, and the FFA kit to measure binding capacity in both the human albumin and FFA‐free bovine albumin. Lastly, we mixed fresh human whole blood 4:1 with PBS, human albumin, FFA‐free bovine serum albumin (BSA), or BSA that was first saturated to its binding capacity with OA and measured the amount of hemoglobin released into the plasma after 30 minute incubation with 0 to 5 mM OA. We found that pharmaceutical‐grade 5% human albumin contained 4.6±0.3 mM FFA (N=5 lots), ~7x the physiologic concentration, leaving an available binding capacity of 4.8±0.6 mM out of a total binding capacity of 9.4±0.8 mM. This represented a significant loss of binding capacity (p<0.0001) compared to the total. FFA‐free BSA (N=5 replicates) had significantly greater available binding capacity of 7.0±1.4 mM (p=0.02) compared to the pharmaceutical‐grade human albumin. In 4:1 mixtures of blood to PBS or albumin, hemolysis was reduced after addition of all concentrations of exogenous OA with 25% FFA‐free compared to 25% pre‐saturated BSA (p<0.04). Likewise, 0.5 and 5 mM exogenous OA caused less hemolysis when blood was pre‐mixed with 5% FFA‐free BSA than with 5% human albumin (p<0.01). These preliminary findings suggest that the failure of albumin in human trials to live up to the promise of animal studies may be due to steps in the manufacturing process that reduce the FFA binding capacity of human albumin.Support or Funding InformationSupported by US Army Medical Research & Materiel Command.