A Study of the Lipids of the Rat Aorta During Induced Calcification

Abstract

Gilman et al. (1) produced calcification in the aortas of rats by administering massive oral dosages of vitamin D₂. This model of induced ectopic calcification has been used by Fleisch et al. (2), Irving et al. (3) and others to study factors controlling the calcification process. As had been consistently observed before in normal calciflying tissue (4–7), Irving et al. (3) found that calcified areas of the rat aortas, so produced, were strongly Sudan black positive, whereas normal aortas were only weakly stained. This model system seemed useful for the study of lipids in calcifying tissue because calcification could be induced under carefully controlled conditions. Any obvious changes in the lipid composition accompanying the aortic calcification should be of significance in elucidating the role of lipids in the calcification process.

Materials and Methods. Two experiments were performed in which 150–200 g female rats of the Wistar strain were given large oral dosages of vitamin D_>3 for 5 consecutive days, the control groups receiving none. The animals were sacrificed on the fourth day after the final dosage of vitamin _D3. In the first experiment 75,000 IU of vitamin _D3/kg of body weight per day were given; in the second experiment 75,000 IU/rat per day were administered. The aortas (from the aortic arch to the bifurcation into the iliac arteries) were dissected free, collected in a solution of 0.9% NaCl at 4°, carefully cleaned of adhering tissue, split lengthwise, rinsed three times with fresh cold saline, blotted free of excess moisture, weighed. Lipids were extracted from the pooled fresh tissues with choloroform-methanol (8) using five consecutive extractions, the first of which lasted overnight; the rest allowing 1–2 hr contact between tissue and solvent.