Age and Sex- Related Changes in Cholesterol Metabolism in Rats Šošić-Jurjević B1., Lütjohann D2., Filipović B1., Vojnović Milutinović D1., Trifunović S1., Jarić I1., Tanić N3., Milošević V1. 1Institute for Biological Research „Siniša Stanković“, University of Belgrade, Belgrade, Serbia; 2Institute for Clinical Chemistry and Clinical Pharmacology, University Clinics, Bonn, Germany; 3) Vinča Institute of Nuclear Sciences, University of Belgrade, Serbia Introduction Materials and Methods Experimental design Wistar rats were housed in the unit for experimental animals at the Institute for Biological Research "Siniša Stanković", Belgrade, Serbia. All animals were fed standard (Veterinarski zavod, Subotica, Serbia) ad libitum and were maintained at constant light (12h light/12h dark) and temperature (21 ± 2°C) conditions. At 4 and 24 months of age, rats (n = 6 per experimental group) were killed by decapitation. Their livers were perfused with cold physiological saline buffer. Blood was collected from the trunk, centrifuged and serum was separated from the blood clot. Serum and liver sampes were stored at – 70 °C for subsequent sterol analysis. Preparation of tissue samples Prior to analysis, the tissue samples were spun in a speed vacuum dryer for 24 h at room temperature in order to express the individual sterol concentrations relative to the dry weight. The sterols were extracted from the dried tissue by placing in a 1.5 ml mixture of chloroform/ methanol (at a 3:1 ratio) for 24 h at 4 C. Hypercholesterolaemia and cardiovascular disease (CVD) are strikingly more common in men than in women. However, in both sexes serum cholesterol levels increase with age, as does the incidence of CVD. The mechanisms responsible for the age-related hypercholesterolemia are still not well understood. Some evidence demonstrates that the causes of age-related disruption of lipid homeostasis include, besides other mechanisms, the gradual decline in ability to remove cholesterol through conversion to bile acids, and the decreased activity of the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7α-hydroxylase. To the best of our knowledge, no one has studied so far the age- and gender- related differences in concentration of cholesterol precursors, as well as its oxidative degradation metabolites in liver and sera of rat animal models, or changes in phytosterol concentrations in sera as putative markers of changes in hepatic and intestinal cholesterol absorption and elimination. Sterol extraction and analyses After alkaline hydrolysis and extraction by cyclohexane serum and hepatic sterols and oxysterols were silylated prior to gas chromatographic (GC) separation. Concentrations of cholesterol were measured by GC-flame ionization detection using 5 α -cholestane as internal standard. Cholesterol precursors such as lathosterol, lanosterol, and desmosterol, and the phytosterols - campesterol and sitosterol - were determined by GC-mass spectrometry-selected ion monitoring, using epicoprostanol as internal standard. The cholesterol metabolites and bile acid precursors 24- and 27-hydroxycholesterol were quantified using [ 2H4]24- and [2H5]27-hydroxycholesterol as internal standard (isotope dilution methodology). Statistical Analyses Statistical analyses of all obtained results were performed using GraphPad Prism 6. Normality of distribution was tested by Kolmogorov-Smirnov test, while the equality of variance was tested by Bartlett’s test. After confirmation of Gaussian distribution and homogeneity of variance, results were subjected to two-way analysis of variance (ANOVA) and then to Bonferroni’s multiple comparison test. Statistical significance was set at p<0.05. The data are presented as the means ± SD. Results Fig. 1 Age- and gender-related changes of cholesterol and related sterols Fig. 2 Age- and gender-related changes of cholesterol metabolites Fig. 3 Age- and gender-related changes of phytosterols H1.2 Oxyphytosterol plasma concentrations H2.2 Oxyphytosterol valve cusp tissue concentrations Discussion and Outlook - Female rats have lower serum cholesterol compared to males (effect of gender, p<0.05; two-way ANOVA). They also have higher concentrations of 7α - and 27-hydroxycholesterol (major intermediary catabolites in degradation of cholesterol to bile acids) both in liver and in sera (effect of gender, p<0.05; two way ANOVA). Hepatic cholesterol levels remain similar between sexes. Desmosterol, the precursor of cholesterol, is higher in young-aged females compared to young-aged males (effect of gender, p<0.005; two way ANOVA). The data indicate that serum cholesterol levels in female rats, in comparison to males, is more likely to be lower due to higher catabolism of cholesterol to bile acids in females, and not due to differences in hepatic biosynthesis of cholesterol. - Age-related increase in serum cholesterol levels, which is detected in both gender (effect of age, p<0.005, two-way ANOVA), was not associated with changes in hepatic cholesterol levels. Moreover, in both gender we detected age-related increase in hepatic 27-hydroxycholesterol (effect of gender, p<0.05; two-way ANOVA) and decrease in hepatic desmosterol (effect of gender, p<0.05; two way ANOVA). Obtained results strongly indicate that age-related increase in serum cholesterol in rats is generally neither a result of changes in hepatic cholesterol metabolism, nor hypothesized decrease in hepatic catabolism and production of 7α- hydroxycholesterol. - Age-related increase in serum phytosterols was detected only in female rats (effect of age, p<0.05; two way ANOVA), which may be a consequence of increased hepatic and extrahepatic absorption and/or decreased elimination of phytosterols. Assumed mechanisms may also contribute to the detected increase in serum cholesterol levels in female rats. - On the other hand, in males, age-related increase in concentration of cholesterol precursors in sera (effect of age, p<0.05; two way ANOVA), namely lanosterol and desmosterol, indicate increased whole body synthesis of cholesterol, which may not be detected in liver due to rapid metabolism of cholesterol in this organ. -Further determinations of concentration of examined sterols and cholesterol metabolites in the intestine (besides liver most important organ in cholesterol absorption, synthesis and elimination) both in females and in males are needed to confirm our assumptions.   Acknowledgments The project was supported by grant from the Serbian Ministry of Education, Science (No. 173009). B. Š. Jurjević was supported by European Society for Endocrinology (ESE) Short-Term Fellowship.