###### Abstract  
Trimethylamine (TMA) is an aliphatic tertiary amine produced by gut microbiota, starting from dietary precursors such as L-choline, L-carnitine and betaine. TMA and its metabolite trimethylamine-N-oxide (TMAO) are elevated in the plasma of cardiovascular disease (CVD) patients. Despite extensive literature on this topic, the scientific community is still divided on which of the two molecules is responsible for the harmful effects on human health. To assess whether the plasma levels of these molecules are also modulated by interactions with macromolecules present in the plasma, the weak bonds between TMA or/and TMAO with human serum albumin (HSA) were studied via molecular docking and spectrofluorimetric assay. The impact of TMA and TMAO on HSA and low-density lipoproteins (LDL) oxidation was also evaluated. Docking analysis shows three main binding sites for TMA and two for TMAO. Spectfluorimetric results show interactions of HSA with TMA and TMAO; a significant (\(p\) = 0.010) decrease in Trp-214 intrinsic fluorescence of HSA was measured starting from the lowest concentrations of both TMA and TMAO (3.26 nM and 29.2 nM, respectively). Furthermore, at all concentrations tested, no significant effect on the formation of carbonyls in HSA was measured (\(p\) > 0.05) in the presence of TMA or TMAO. However, 28.6 mM TMAO significantly increased (\(p\) < 0.05) the degree of oxidation of LDL, suggesting that TMAO has a pro-oxidant role on LDL. H +
Footnote †: journal: _Sci._**2023**, 13, 421. [[https://doi.org/10.3390/app13010421](https://doi.org/10.3390/app13010421)]([https://doi.org/10.3390/app13010421](https://doi.org/10.3390/app13010421))  
Footnote †: journal: _Sci. determine if and which conformational changes are induced by a particular molecule or chemical species in cases where it binds to HSA. In fact, the intensity of Trp fluorescence depends on the surrounding environment; in particular, if Trp is in an apolar environment and is exposed to a solvent or forms hydrogen bonds, its emission spectrum shifts towards higher wavelengths and its quantum fluorescence yield (\(\Phi\)F) decreases; moreover, the fluorescence intensity can increase or decrease according to the specific microenvironment and the presence of quenching groups. HSA also contains 35 Cys residues, 34 of which form intramolecular disulphide bridges that significantly determine the stability and the relatively long half-life of the protein (around 20 days) [1]. The remaining residue of Cys in position 34 represents the most abundant thiol group in plasma; for about 75%, it is in a reduced form, consequently playing a crucial role as an antioxidant in the blood. The reactivity of this residue is limited by the steric hindrance provided by the surrounding environment; this prevents the formation of intermolecular disulphide bridges between two albumin molecules, a phenomenon that could cause negative effects due to the consequent lowering of oncotic pressure. Trimethylamine (TMA) is an aliphatic tertiary amine produced by gut microbiota, starting from precursors present in the diet such as L-choline, L-carnitine and betaine [5]. Choline can be present in free or esterified form, thus having different ways of absorption in the intestine; the form most converted to TMA is mainly the free one; carnitine and its metabolite \(\gamma\)-butyrobetaine also derive from the consumption of animal products, such as meat, fish and milk, while betaine is contained mainly in vegetables such as spinach, beets and cereals [6]. Starting from these precursors, intestinal bacteria produce TMA via TMA lyases; trimethylamine oxide (TMAO) is generated in the liver by flavin-monooxygenases (FMOs) to serve as the final form and this is eliminated through the kidney [7]. A positive correlation between elevated plasma TMAO and an increased risk for major adverse cardio and cerebrovascular events suggests that TMAO can contribute to CVD aetiology [8; 9].