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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Enzyme blockade: a nonradioactive method to determine the absolute rate of cholesterol synthesis in the brain.

The standard in vivo method to determine rates of brain cholesterol synthesis involves systemic injection of (3)H(2)O and measurement of incorporated radioactivity in 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 [(3)H]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 with 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 approximately 8 h) for 3 days. The rate of brain cholesterol synthesis determined by this method ( approximately 30 microg/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 rate and cerebellum having the lowest rate.[1]


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