Natural Polyamine Spermidine Inhibits the In Vitro Oxidation of LDL

Abstract

Spermidine is a natural autophagy-inducer and anti-aging compound. Herein, we investigated a potential autophagy-independent mechanism of spermidine, namely its capability to directly impede LDL oxidation, an early step in atherogenesis. In our in vitro-model, LDL oxidation was induced by the addition of CuCl₂ in the presence of increasing concentrations of spermidine, and the degree of oxidation of the lipid, as well as of the protein part of LDL, was measured. We found that spermidine concentration-dependently inhibited the production of lipid hydroperoxides, malondialdehyde, and oxidation-specific immune epitopes in the LDL particle, associated with decreased relative electrophoretic mobilities, respectively. For example, the LPO content was significantly lower when LDL was oxidized in the presence of 500 µg/mL spermidine (26.9 ± 1.6 nmol/mg LDL) than in the absence of spermidine (180.6 ± 7.7 nmol/mg LDL, p < 0.0001). When oxLDL was obtained under increasing spermidine concentrations, its cytotoxicity in EA.hy926 cells concentration-dependently decreased. Quantum chemical calculations show that the reaction between spermidine and hydroxyl radicals is exergonic. We conclude that spermidine is a direct inhibitor of LDL oxidation due to its capability to scavenge hydroxyl radicals. Thus, spermidine supplementation might be a suitable tool to impede atherogenesis and associated (cardio)vascular diseases. Further prospective clinical studies are needed to evaluate the potential atheroprotective/health-promoting effects of spermidine-rich diets.

Keywords: antioxidants; atherogenesis; copper ions; endothelial damage; lipid oxidation.

Figure 1

Cu²⁺-induced oxidation of the LDL particle’s lipid component. (A) Spermidine significantly (p < 0.0001, ANOVA with Bonferroni post-test) suppressed LPO formation; (B) Spermidine significantly (p < 0.0001, ANOVA with Bonferroni post-test) suppressed MDA formation after 4 h of incubation time. Data represent mean ± SD from four separate measurements; *… p ≤ 0.05, **… p ≤ 0.01, ***… p ≤ 0.001.

Figure 2

Cu²⁺—induced oxidation of the LDL particle’s protein component. (A) Spermidine significantly (p < 0.0001, ANOVA with Bonferroni post-test) suppressed the formation of oxidation-specific epitopes on the LDL particle; (B) spermidine significantly (p < 0.0001, ANOVA with Bonferroni post-test) reduced REM values after 4 h incubation time. Data represent mean ± SD from four separate measurements; **… p ≤ 0.01, ***… p ≤ 0.001.

Figure 3

Suppression of LDL oxidation by spermidine vs. alpha-KG. (A) LPO formation in the absence (spheres) and in the presence of 250 µg/mL (squares) or 500 µg/mL (triangles) of alpha-KG; (B) LPO formation in the absence (spheres) and in the presence of 250 µg/mL (squares) or 500 µg/mL (triangles) of spermidine. Two typical experiments are shown.

Figure 4

Spermidine during LDL oxidation reduces the cytotoxic effects of oxLDL. nLDL was preincubated with 0, 50, or 100 µg/mL spermidine and subsequently oxidized with CuCl₂ until the LPO content without spermidine reached 100 nmol/mg LDL protein. The EAhy.926 cells were then incubated with the thus obtained oxLDLs (0.4 mg/mL). Cell viability concentration-dependently increased with oxLDLs formed under increasing levels of spermidine. Data represent mean ± SD from four separate measurements; ***… p ≤ 0.001. Cell viability values for incubation with nLDL was set to 100% for each experiment.

Figure 5

Chemical structure of spermidine. Large dark spheres: nitrogen; large light spheres: carbon; small spheres: hydrogen.