The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

Abstract

The preservation for EM of saturated phospholipids [from rats] in general and phosphatidyl choline (PC) in particular, remains an unsolved problem since OsO4 and glutaraldehyde are incapable of interacting with PC directly. By introducing tannic acid preceding osmication, highly ordered, preserved lamellar structures were demonstrated in model experiments with saturated PC and in vivo experiments with type II pneumocytes of lung tissue. The secretory bodies of the latter contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 .ANG.. Tannic acid apparently interacts with the choline component of PC to form a complex, which then could be stabilized by treatment with OsO4. In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contain choline, behaved similarly in model experiments. There was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS) or phosphatidyl inositol (PI). Chemical studies indicated a high pH dependency for the formation of the PC-tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate, leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO4 and lead citrate stains.