Abstract
Protein aggregation and amyloid fibril formation are key events in neurodegenerative disorders such as Alzheimer's disease, and are worsened by non-enzymatic glycation, which destabilizes protein structure. Glycation by reducing sugars like glucose produces advanced glycation end products (AGEs), promoting misfolding and aggregation. β-Hydroxybutyrate (BHB), a ketone body with anti-glycation and neuroprotective properties, may counteract these effects. This study examined structural, aggregation, and fibrillar changes of human serum albumin (HSA) after prolonged glucose-induced glycation, and evaluated the modulatory role of BHB via a multi-technique approach. HSA was incubated for 120 days under four conditions: control, BHB alone, glucose alone, and glucose + BHB. Analyses included atomic force microscopy (AFM), circular dichroism (CD) spectroscopy, ANS fluorescence, Thioflavin T (ThT) fluorescence, and Congo Red assays. AGE formation was quantified to link biochemical modifications with aggregation patterns. Glucose treatment markedly increased AGE levels. AFM revealed extensive aggregation and high surface coverage in the glucose group, partially reduced by BHB. CD spectroscopy showed α-helix loss and β-sheet enrichment with glycation, while BHB preserved structure. ANS fluorescence indicated glucose-enhanced hydrophobic exposure, reduced by BHB. ThT and Congo Red assays confirmed less amyloid fibril formation in glucose + BHB samples versus glucose alone. These results suggest that glucose induces marked glycation, structural disruption, and amyloidogenic aggregation in HSA, whereas BHB provides partial protection, likely through structural stabilization and aggregation pathway modulation. BHB may offer therapeutic promise for limiting amyloid-related neurodegenerative diseases.