Submitted on May 7, 2004
Revised on July 6, 2004
Accepted on July 13, 2004
Enzyme blockade: A nonradioactive method to determine the absolute rate of cholesterol synthesis in the brain
R. Kennedy Keller, Michael Small, and Steven J. Fliesler
Ophthalmology Dept., Saint Louis University Eye Institute, Saint Louis, MO 63104-1540
Corresponding Author: fliesler{at}slu.edu
The standard in vivo method to determine rates of brain cholesterol synthesis involves systemic injection of 3H2O and measurement of incorporated radioactivity into sterols. Herein, we describe an alternative method (“enzyme blockade”) that obviates the use of radioactivity. The method relies on the ability of AY9944, a potent and relatively selective inhibitor of cholesterol synthesis, to cause the time-dependent accumulation of 7-dehydrocholesterol (DHC), a cholesterol precursor detected with sensitivity and specificity by reverse-phase HPLC-coupled spectrophotometry at 282 nm. To validate the method, adult AY9944-treated and control mice were injected with [3H]acetate. After 24 h, most of the radioactivity in brain sterols from treated mice accumulated in DHC, without significantly perturbing overall sterol pathway activity, compared to controls (where cholesterol was the dominant radiolabeled sterol, with no label found in DHC). When adult mice were treated continuously with AY9944, the time-dependent accumulation of DHC in brain was linear (after ~8 h) for 3 days. The rate of brain cholesterol synthesis determined by this method (~30 
g/g/day) closely agrees with that determined by the radioactive method. We also determined the cholesterol synthesis rate in different regions of adult mouse brain, with frontal cortex having the highest and cerebellum having the lowest rate.
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