(P11-003-23) Diet-Induced Severe Hyperhomocysteinemia Favors Experimental Atherosclerosis Progression and Modifies the Plasma Metabolome

Objectives: Hyperhomocysteinemia is a risk factor for atherosclerosis and vascular disease by incompletely defined mechanisms. We investigated the role that B vitamins play in this disease's progression, metabolically.

Methods: Apolipoprotein E knockout mice, a model for human atherosclerosis that accumulates aortic plaque, were fed ad libitum a control diet (CD), (Kcal %: 70/11/18, carbohydrate/fat/protein; n=8), or a matched diet with decreased levels of vitamins B6, B9, and B12 and enriched in methionine (LMD) (n=16). After 8 weeks, plasma, aorta, and liver were collected for the quantification of methylation metabolites by LC-MS/MS. A targeted metabolic approach by triple quadrupole mass spectrometry was conducted in the plasma. The aortic plaque burden in the brachiocephalic artery (BCA) was quantified by 14T-Magnetic Resonance imaging.

Results: Methylation metabolites were significantly (p < 0.05) altered in the LMD-mice versus controls. These included a severe accumulation of plasma and hepatic homocysteine (plasma: 247 ± 88.1 µM versus 4.7 ± 0.9 µM; liver: 543 ± 285 nmol/g versus 31.8 ± 8.0 nmol/g respectively) that was accompanied by a decreased methylation capacity, assessed by S-adenosylmethionine: S-adenosylhomocysteine ratio, in plasma and aorta, thus revealing the presence of hypomethylating stress intravascularly (plasma: 0.236 ± 0.106 to 0.059 ± 0.041; aorta: 0.571 ± 0.249 to 0.190 ± 0.055). Moreover, a 172-fold increase in aortic cystathionine levels was observed.  Betaine, and its metabolic precursor, choline, were also significantly (p < 0.05) decreased (33 and 1.5 fold respectively) in the liver of LMD mice versus CD-mice, suggesting increased activity of the betaine-dependent remethylation pathway due to the severe accumulation of homocysteine.  High-field MRI analysis revealed significantly (p < 0.05) more atheroma in the LMD mice than in control mice. Targeted metabolomic analysis revealed widespread changes in the plasma metabolome of LMD mice compared to the control, including alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, and triacylglycerol levels.

Conclusions: This study reconfirms the relevance of severe hyperhomocysteinemia to the progression of atherosclerosis and reveals potential biomarkers that warrant further investigation.

Funding Sources: Huck Institutes of the Life Sciences