Metformin is the most commonly prescribed glucose-lowering drug for the treatment of type 2 diabetes. The aim of this study was to investigate whether metformin is capable of impeding the oxidation of LDL, a crucial step in the development of endothelial dysfunction and atherosclerosis. LDL was oxidized by addition of CuCl₂ in the presence of increasing concentrations of metformin. The extent of LDL oxidation was assessed by measuring lipid hydroperoxide and malondialdehyde concentrations, relative electrophoretic mobilities, and oxidation-specific immune epitopes. Cytotoxicity of oxLDL in the vascular endothelial cell line EA.hy926 was assessed using the alamarBlue viability test. Quantum chemical calculations were performed to determine free energies of reactions between metformin and radicals typical for lipid oxidation. Metformin concentration-dependently impeded the formation of lipid hydroperoxides, malondialdehyde, and oxidation-specific immune epitopes when oxidation of LDL was initiated by addition of Cu²⁺. The cytotoxicity of oxLDL was reduced when it was obtained under increasing concentrations of metformin. The quantum chemical calculations revealed that only the reaction of metformin with hydroxyl radicals is exergonic, whereas the reactions with hydroperoxyl radicals or superoxide radical anions are endergonic. Metformin, beside its glucose-lowering effect, might be a suitable agent to impede the development of atherosclerosis and associated CVD. This is due to its capability to impede LDL oxidation, most likely by scavenging hydroxyl radicals.

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.

Background/Objectives: The oxidation of low-density lipoprotein (LDL) is a crucial step in atherogenesis. Beta-Caryophyllene (BCP) is a natural compound with established anti-oxidative and anti-inflammatory properties as shown in animal and cell culture studies. We examined whether BCP can impede LDL oxidation in an in vitro model. Methods: The anti-oxidative effect of BCP was evaluated in different concentrations (0, 25, 50, 100, and 150 µg/mL) with regard to scavenging reactive oxygen species (ROS) during LDL oxidation, which was initiated by the addition of copper chloride (CuCl₂) in a concentration of 10 µmol/L. Lipid hydroperoxides (LPO), malondialdehyde (MDA), dienes, cell viability, and reactions of BCP with ROS according to Gibbs free energies were applied to determine the oxidation state of LDL. Results: Our findings indicated that BCP is highly efficient in inhibiting LDL oxidation in a dose-dependent manner in this in vitro model. The lipid hydroperoxide content in oxLDL was significantly lower in the presence of 100 µg/mL BCP compared to oxLDL without BCP (p < 0.0001). This corresponds to the MDA levels, which were significantly lower in the presence of 100 µg/mL BCP compared to oxLDL without BCP (p = 0.0393). Furthermore, a dose-dependent inhibition of diene formation in the LDL particle was observed in the presence of ascending BCP concentrations which corresponds to a decrease in the cytotoxicity of oxLDL in EA.hy926 cells in the presence of increasing concentrations of BCP. Moreover, BCP’s anti-oxidant effectiveness exceeds that of the widely recognized anti-oxidant spermidine at equivalent concentrations. Our quantum chemical calculations showed that the reactions between BCP and hydroxyl radicals, hydroperoxyl radicals, or hydrogen peroxide are exergonic. We therefore conclude that BCP impedes the oxidation of LDL by its capability to scavenge (at least) these three reactive oxygen species. Conclusions: Our results indicate that BCP impedes the oxidation of LDL in vitro and therefore presumably has the potential to serve as an appropriate therapeutic agent to prevent atherogenesis and related (cardio)vascular diseases by balancing vascular oxidative stress. For this purpose, more prospective clinical studies in humans are required to assess the potential atheroprotective and health-promoting effects of BCP.

Background/Objectives: Fruit, vegetable and berry (FVB) juice powder concentrates are increasingly used to support cardiovascular health. Since they are usually consumed over long periods, this study assessed the metabolic safety of a commercial FVB supplement and its impact on selected biomarkers of systemic oxidative stress and cardiovascular health. Methods: In this open-label, randomized controlled trial, generally healthy older adults were allocated to a control group (n = 16) or an FVB group (n = 18), ingesting six capsules/day. This work reports the pre-specified 6-month timepoint of a 1-year trial. The primary outcome, metabolic safety, was assessed by hepatic, renal, thyroid, lipid and glucose metabolism markers. Exploratory analysis assessed carbonyl proteins, malondialdehyde, and oxidized LDL as markers of systemic oxidative stress, together with homocysteine and blood pressure as markers of cardiovascular health, while plasma vitamin C served as a compliance marker. Results: The FVB concentrate was well tolerated, and no safety-relevant deteriorations were observed in the FVB group. Carbonyl proteins decreased significantly in the FVB group (interaction p = 0.030). Homocysteine remained stable in the FVB group but increased significantly in the control group (interaction p = 0.002). Furthermore, systolic blood pressure decreased significantly (interaction p = 0.036), with a comparable interaction of diastolic blood pressure (p = 0.011), and plasma vitamin C increased significantly in the FVB group (interaction p = 0.004). After adjustment for baseline values, age and BMI, between-group differences persisted for homocysteine and diastolic blood pressure, whereas the differences in systolic blood pressure and carbonyl proteins were attenuated and no longer reached statistical significance. Conclusions: Six months of supplementation with a commercial FVB concentrate was metabolically safe and well tolerated. Since these findings are based on a pre-specified timepoint and a limited number of participants, they are hypothesis-generating rather than confirmatory and require confirmation by the complete trial. Registered at ClinicalTrials.gov (NCT04763291; registered on 21 February 2021).

Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation, yet its relationships with lipoprotein subclasses remain poorly explored, particularly in metabolic syndrome (MS). We investigated associations of free MDA measured by the TBARS assay (MDA-TBARS) and total MDA quantified by high-performance liquid chromatography (MDA-HPLC) with NMR spectroscopy-measured lipoprotein subclasses in 65 healthy volunteers (HV) and 65 patients with MS. MDA-HPLC concentrations were higher than MDA-TBARS, and the two measures were strongly correlated. Free MDA levels were significantly elevated in MS, whereas total MDA did not differ between groups. In HV, MDA-TBARS showed positive relationships with VLDL and IDL, as well as triglyceride enrichment of LDL, IDL, and HDL, and a negative relationship with levels and cholesterol content of small, dense HDL, as well as cholesterol and phospholipid content of LDL subclasses. MDA-HPLC only showed a positive association with VLDL levels in HV. In contrast, no significant associations between MDA and lipoproteins were observed in MS. Multivariable analyses confirmed that lipoprotein profiles were able to discriminate between low and high MDA levels in HV, but not in patients with MS. In conclusion, interactions between oxidative stress and lipoproteins are strongly context dependent, being preserved in health but disrupted in MS, with free MDA emerging as a more informative marker of lipoprotein-associated lipid peroxidation.