Fig. 3.

Cysteamine inhibits LDL oxidation by iron at pH 4.5 and ceroid formation in macrophages.

(A) LDL (50 μg of protein/ml) in NaCl/sodium acetate buffer (pH 4.5) was incubated with 5 μM FeSO₄ at 37 °C in quartz cuvettes. Cysteamine (final concentrations 25, 50 and 250 μM) was added to the cuvettes at the start of the incubation. Oxidation and UV scattering were monitored by measuring the change in attenuance (absorbance plus UV scattering) at 234 nm against appropriate reference cuvettes. The stages of oxidation are marked with arrows: 1, lag phase; 2, rapid oxidation phase; 3, slow oxidation phase; 4, aggregation phase and 5, sedimentation phase. These data are representative of three independent experiments. Cysteamine at 25 μM increased the time required to increase the attenuance to 0.1 by 5.5 ± 1.5 fold (p < 0.05) and at 50 μM by 14.4 ± 1.2 fold (p < 0.001), mean ± SEM of 3 independent experiments; ANOVA and Dunnett's post-hoc test. (B) Human monocyte-derived macrophages were cultured on coverslips and incubated with SMase-LDL at 200 μg protein/ml for 24 h. They were washed and cultured for 7 days with RPMI 1640 medium containing 10% (v/v) human lipoprotein-deficient serum to which cysteamine was added every 24 h. The cells were then washed, fixed, treated with ethanol and xylene to remove non-ceroid lipids and stained for ceroid with Oil Red O. Ceroid was quantified using ImageJ. (C) Mean ± SEM of 4 independent experiments. *p <0.001, ANOVA and Dunnett's post hoc test.