Increased F2-isoprostanes in liver and colon of rabbits fed a high cholesterol diet. Influence of Fe and Zn supplementation

Abstract

We have been investigating an animal hypercholesterolemia animal model to examine different influences on atheroma formation and tissue damage. Rabbits were fed 1% cholesterol for 8 weeks. After 3 weeks animals were supplemented with either desferal (72 mg/kg), Zn (1 g/kg) or vehicle by daily s.c. injection for 5 days/week. Tissues were immediately freeze clamped and then lipids extracted using the Folch extraction. Total F2-isoprostanes (free+esterified) were measured by GC-MS with NICI after lipid hydrolysis, followed by a single anion exchange solid phase extraction (AnExSPE) procedure developed in our laboratory and finally two derivatization steps. The AnExSPE procedure is able to sufficiently purify the F2isoprostane fraction for GC-MS (NICI) analysis without the need for HPLC or TLC and provides accurate quantitation using two different deuterated F2-isoprostanes. Recovery of F2-isoprostane was calculated to be 49.877.9% and 44.678.6% (mean7SD) in liver and colon, respectively. Sensitive analytical quantitation was verified by spiking Folch extracts with F2-isoprostane standard. Control tissue levels of F2-isoprostanes in rabbit liver and colon were 1.370.3 and 0.670.1 ng/g tissue respectively (mean7SD, n ¼ 5 animals), which is similar to values reported for rodent tissue [1]. Cholesterol-fed rabbits had significantly elevated F2-isoprostanes in liver and colon compared to rabbits fed a normal diet. Supplementation with desferal had no significant effect on the cholesterol-induced increase in colon and liver. However, treatment with Zn protected against the F2-isoprostane increase in both tissues studied. Our results indicate that damage to tissue lipids by a high-cholesterol diet may be significantly influenced by levels of metal ions in the diet. We are currently examining plasma and other tissues in this rabbit model of atheroslerosis, to investigate F2-isoprostanes as a sensitive biomarker of lipid peroxidation.