(755b) Understanding Atherogenesis Using Colloid Science: Effect of Sphingomyelinase-Mediated Generation of Ceramide on Aggregation of Low Density Lipoproteins

This study addresses the
Response-to-Retention hypothesis, which states that the subendothelial
retention of atherogenic lipoproteins is the necessary and sufficient condition
for the initiation of atherosclerosis.  Here we focus on the relationship
between the generation of ceramide in the low density lipoprotein (LDL)
phospholipid monolayer and the resulting aggregation of LDL particles.  This
study provides the first measurement of neutral, Mg²⁺-dependent
Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and
does so for a range of enzyme concentrations (0-0.22 units Smase/mL).  The
kinetics of ceramide generation was measured using a fluorescence assay for the
above enzyme concentrations with a fixed substrate concentration (0.33 mg
LDL/mL).  The kinetics of LDL aggregate formation was measured by dynamic light
scattering (DLS, method of cumulants) for identical enzyme concentrations. 
Ceramide concentration profiles were fit with a modification of the Michaelis-Menten
model (k_a=1.11 x 10^-1 µM^-1min^-1, k_-a=6.54
x 10² µM^-1min^-1, k₁=3.33 x 10¹
µM^-1min^-1, k_-1=1.41 x 10^-2 min^-1,
k_cat=8.05 x 10¹ min^-1, K_M=2.418 µM,
k_deact=4.66 x 10^-2 µM^-1min^-1) that
accounts for the effects of enzyme attachment to the LDL monolayer and for
deactivation of Smase due to product inhibition.  LDL aggregation is described
by a Mass Action Model as explained in previous studies.  A key result of this
work is the finding that LDL aggregate size depends directly on ceramide
concentration and is independent of enzyme concentration.  This study
demonstrates how principles of colloid science are relevant to important
biomedical problems.