A comparative study of the accurate measurement of endotoxin in liposome-encapsulated hemoglobin

We have examined three different methods of endotoxin determination utilizing the Limulus Amebocyte Lysate (LAL) assay to accurately determine endotoxin levels in Liposome Encapsulated Hemoglobin (LEH), 1) the gel-clot method, 2) chromogenic spectroscopic-based LAL, and 3) the turbidimetric method which determines endotoxin levels in solutions based on the time needed to reach a specific degree of turbidity. Both the chromogenic and turbidimetric methods require significant dilution of the LEH preparation before accurate measurement can be made. We have tested the levels of endotoxin in LEH solutions using these methods and measured LEH, liposome, and hemoglobin samples spiked with known amounts of endotoxin. A comparison of the three methods shows that the absolute value of endotoxin measured in LEH by the three methods can vary significantly. However, within any one assay the spiked amount of endotoxin in the sample can be accurately measured. The accuracy of these methods may also be complicated by the binding of endotoxin to LEH. This was evident by mixing free endotoxin with LEH followed by centrifugation to separate the LEH. Biological activity of endotoxin bound to LEH was measured by exposure to RAW264.7 followed by the expression of tumor necrosis factor.