A Freeze-fracture TEM and Small Angle X-ray Diffraction
Study of the Effects of Albumin, Serum and Polymers on
Clinical Lung Surfactant Microstructure
Andreas Braun 1, Patrick C. Stenger 1, Heidi E. Warriner 1, Joseph A. Zasadzinski 2*, Karen W. Lu 3 and H. William Taeusch 3
1 UC Santa Barbara
2 Univ. of California
3 UC San Francisco
* To whom correspondence should be addressed. E-mail: gorilla{at}engineering.ucsb.edu.
Submitted on August 21, 2006
Revised on January 18, 2007
Accepted on 28 February 2007
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Abstract |
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Freeze-fracture transmission electron microscopy (FFTEM) shows significant differences in the bilayer organization and fraction of water within the bilayer aggregates of clinical lung surfactants, which increases from Survanta to Curosurf to Infasurf. Albumin and serum inactivate all three clinical surfactants in vitro; addition of the nonionic polymers polyethylene glycol (PEG), dextran, or hyaluronic acid (HA) also reduces inactivation in all three. FFTEM shows that PEG, HA and albumin do not adsorb to the surfactant aggregates, nor do these macromolecules penetrate the interior water compartments of the surfactant aggregates. This results in an osmotic pressure difference that dehydrates the bilayer aggregates, causing a decrease in the bilayer spacing as shown by small angle X-ray scattering and an increase in the ordering of the bilayers as shown by freeze-fracture electron microscopy. Small angle X-ray diffraction shows that the relationship between the bilayer spacing and the imposed osmotic pressure for Curosurf is a screened electrostatic interaction with a Debye length consistent with the ionic strength of the solution. The variation in surface tension due to surfactant adsorption measured by the pulsating bubble method shows that the extent of surfactant aggregate reorganization does not correlate with the maximum or minimum surface tension achieved with or without serum in the subphase. Albumin, polymers and their mixtures alter the surfactant aggregatemicrostructure in the same manner, hence, neither inhibition reversal due to added polymer nor inactivation due to albumin is caused by alterations in surfactant microstructure.
Key Words:
electron microscopy, freeze-fracture, inhibition, pulmonary surfactant, small angle X-ray scattering
