At various postnatal days, tissues (e.g., serum, liver, retina,
brain) from treated and control pups (two to four animals per time
point; three experiments) were harvested and saponified, and the
nonsaponifiable lipids were extracted and analyzed by reversed-phase
high-performance liquid chromatography (HPLC) to quantify the types and
amounts of sterols. Methods used were essentially those described
previously,
15 except for the commercial system used:
reversed-phase column, 150 × 4.6 mm (model IB-SIL 3 C18 BDS;
Phenomenex, Torrance, CA); guard column (mobile phase, MeOH at 1
ml/min; detection at 205 nm; NovaPak C18; Waters/Millipore, Milford,
MA). Using this system, retention times (relative to those of
cholesterol (Δ
5, 1.00 minute) for the following authentic
isoprenoid lipid reference standards were obtained:
cholesta-5,7,24-trien-3β-ol (Δ
5,7,24), 0.70 minutes;
cholesta-5,24-dien-3β-ol (desmosterol, Δ
5,24), 0.79
minutes; cholesta-5,8-dien-3β-ol (8-dehydrocholesterol,Δ
5,8), 0.83 minutes; cholesta-5,7-dien-3β-ol
(7-dehydrocholesterol, Δ
5,7), 0.89 minutes; squalene,
1.21 minutes. HPLC peak assignments were made in comparison with these
reference standards, and each sterol was quantified by integrated peak
area analysis in comparison with the empirically determined response
factor (integration units per nanomole) for the given standard
compound. The relative response factors (relative to cholesterol, 1.00)
were as follows: cholesta-5,7,24-trien-3β-ol, 2.78; desmosterol,
2.53; 7-dehydrocholesterol, 1.10; squalene, 12.89.