P37 Changes in the hydrogen sulfide (H2S) system arising from administration of high fat diet

Abstract

Atherosclerosis is a progressive disease characterized by endothelial dysfunction and accumulation of lipids, cellular debris and fibrous elements within the intima of the vessel wall. Eventually, atherosclerosis triggers plaque formation, vascular remodeling and luminal obstruction [1] . It is becoming increasingly clear that atherosclerosis is an inflammatory disease, dependent on the interaction between inflammatory cells recruited in response to endothelial lipid accumulation, and to the local, wound-healing response of surrounding vascular smooth muscle cells. H 2 S is a gasomediator synthesized from cysteine predominantly by cystathionine- γ -lyase (CSE) [2] and been reported to exhibit both pro- and anti-inflammatory effects [3] . Administration of high fat diet over an extended period results in the development of a low grade inflammatory response which can predispose to atherosclerosis. In the present work we therefore investigated the role of H 2 S in high fat (HF) fed mice. Mice (C57/Black, male, 25 g) were fed either normally or given a HF diet (16% fat, 12.5% cholesterol and 5% sodium cholic acid) for up to 16 weeks. At the end of 8, 12 or 16 weeks, groups of mice were anaesthetised, blood obtained by cardiac puncture and centrifuged for plasma and heart, liver, kidney and lungs removed for biochemical and histological analyses. H 2 S synthesizing enzyme activity was measured in lung, kidney and liver homogenates incubated with L-cysteine (10 mM) and pyridoxal 5′-phosphate (2 mM) by the zinc trapping spectrophotometric assay. Plasma H 2 S levels were determined by high-performance liquid chromatography (HPLC) [4] . Lung, kidney and liver homogenates were used for western blotting of cystathionine- β -synthetase (CBS), CSE and 3-mercaptopyruvate synthase (3-MST). A range of cytokines and chemokines were also assayed in mouse plasma using a Bio-Plex Pro ™ Assay. Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits. Oil red O staining was performed on aorta samples. Compared to normal diet mice, HF mice showed similar H 2 S synthesizing activity for lung (normal mice 0.28 ± 0.04 nmol/mg, n = 7; HF mice 0.22 ± 0.03 nmol/mg, n = 7), and decreased H 2 S synthesizing activity for kidney (normal mice 2.49 ± 0.79 nmol/mg, n = 7; HF mice 1.16 ± 0.32 nmol/mg, n = 7) and liver (normal mice 9.21 ± 2.44 nmol/mg, n = 7; HF mice 1.65 ± 0.34 nmol/mg, n = 7) (p 2 S levels (normal mice 256.7 ± 18.4 nM; HF mice 274.6 ± 17.1 nM, n = 5). For western blots, HF fed mice showed increased CBS in liver and kidney, decreased CSE in liver and lung, decreased 3-MST in liver, but similar expression of 3-MST in kidney and lung. HF fed mice had elevated plasma levels of IL-12 (p40), G-CSF, IL-6 and MCP-1. There was no difference between groups in terms of plasma SAA and CRP. There was little difference in aortic lipid content between groups as assessed histologically by oil red O staining. Taken the data together, we propose that changes in H 2 S metabolism are apparent in these animals prior to the development of frank atherosclerosis. Thus, it is possible that monitoring H 2 S may be useful as a predictive biomarker for atherosclerosis.