Ilenia Lorenza Calcaterra (1), Guido D'Errico (1), Carmine De Luca (1), Martina Ferrandino (2), Maria Donata Di Taranto (2), Giuliana Fortunato (2), Gabriella Iannuzzo (3), Alessandro Di Minno (4), Matteo Di Minno (3)

(1) Dipartimento di Medicina Clinica e Chirurgia, Università di Napoli, Ferderico II.
(2) Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II.
(3) Dipartimento di Medicina Clinica e Chirurgia, Università di Napoli, Federico II.
(4) Dipartimento di Farmacia, Università di Napoli, Federico II.

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Background: PCSK9 inhibitors (PCSK9i) provide robust LDL-C reduction in familial hypercholesterolemia (FH). Their effects on homocysteine (Hcy), vascular biomarkers, and metabolic pathways relevant to methylation remain incompletely characterized.

Methods: We enrolled FH consecutive patients treated with Evolocumab for 12 weeks on top of standard therapy. Lipid profile, plasma Hcy, oxidative-stress and endothelial biomarkers (isoprostanes, DH series, ADMA, SDMA, arginine bioavailability) were measured at baseline and follow-up. Paired t-tests quantified within-patient changes; multivariate linear regression identified independent predictors of ΔHcy. Metabolomic signals were examined for pathway coherence with Hcy metabolism.

Results: We included 25 FH patients. LDL-C decreased by 49% (from 201 ± 70 to 103 ± 58mg/dL), total cholesterol by 35%, and Lp(a) by 17%. Plasma Hcy fell by 29% (from 12.6 ± 6.7 to 8.9 ± 2.4 µmol/L, p<0.001). Oxidative stress improved: isoprostanes −15.3% (p=0.017) and DH2 −17.2% (p=0.045); DH1 showed a trend (−11.7%, p=0.072). Endothelial markers showed non-significant mean changes (ADMA +5.3%, p=0.37; SDMA −9.1%, p=0.21; arginine bioavailability −8.2%, p=0.13). Correlation analyses linked larger ΔHcy reductions with greater improvements in LDL particle indices and Lp(a). In multivariate analysis, the change in LDL score (ΔLDL score) emerged as the strongest independent predictor of ΔHcy (β −0.028, p<0.001) while SDMA, ADMA, arginine bioavailability, isoprostanes, and ΔLp(a) contributed as additional, smaller predictors. Metabolomic read-outs indicated lower plasma choline and modulation of the betaine-dependent remethylation pathway of Hcy, providing a mechanistic link between lipid changes, methylation flux, and Hcy reduction.

Conclusions: Beyond LDL-C lowering, PCSK9i therapy is associated with a 29% reduction in Hcy and measurable improvements in oxidative stress. ΔLDL score is the key predictor of Hcy change, supporting a tight relationship between LDL particle characteristics and homocysteine metabolism. The concurrent metabolomic signature (choline decrease; betaine-pathway engagement) reinforces a pleiotropic vascular benefit of PCSK9i in FH. This research was supported by the European Union PNRR-MAD-2022-12375721 – CUP C63C22001520006 – NRRP M6C2I2.1 NextGenerationEU.