## 4 Discussion  
Variations in plasma levels of TMA and its metabolite TMAO are associated with CVD, thus representing a potential biomarker for the prevention of these diseases; however, it is still unknown which of the two compounds (if any) is decisive as a risk factor for CVD. Previous studies have reported that the plasma level of TMA is slightly higher in men than in women, while that of TMAO is similar in the two genders [22]. By comparing two groups of people, the first made up of vegetarians and vegans, the second made up of omnivores, it was found that the plasma levels of TMAO were significantly lower in the first group [23]. Moreover, a reduced ability to produce TMAO starting from carnitine was noted in the first group, probably because of the reduced ability of the microbiota of the vegetarian and vegan subjects to convert carnitine into TMA; this finding suggests that dietary habits have a significant impact on plasma levels of TMA and TMAO [23]. Moreover, due to its low molecular weight (75.1 Da), TMAO is easily excreted in the kidney. Studying the renal excretion of radioactive isotope-labelled TMA and TMAO, it has been shown that both compounds are eliminated by approximately 95% within the first 24 h. However, while TMA is mostly eliminated within the first 3 h following exponential  
Figure 4: LDL oxidation was measured by a change in absorbance (mean value \(\pm\) SD) at 234 nm of LDL (0.01 mg/mL) incubated with 5 \(\mu\)M Cu\({}^{++}\) with (**A**) 0.6 \(\mu\)M TMA, 5.37 \(\mu\)M TMAO; (**B**) 0.82 mM TMA, 9.54 mM TMAO; (**C**) 2.46 mM TMA, 28.6 mM TMAO; (**D**) 24.6 mM TMA, 2.86 mM TMAO; \({}^{*}p<0.05\). dynamics, the elimination of TMAO presents as constant kinetics during the first 9 h after administration [23]. Physiologically, TMAO is an osmolyte that acts as a stabilizer of proteins. It helps to maintain correct protein folding, counteracts the effect of denaturants such as changes in pH, urea and high pressure, and as such, is important for maintaining the enzymatic activity of the enzymes to which it binds [24]. Recently, numerous studies have examined TMAO and its precursor TMA for their potential role in the onset of CVD [11; 12; 13; 14; 15; 25; 26; 27; 28]. However, considering the literature and the present research, the results remain contradictory; although some studies suggest that TMAO produces harmful effects on the cardiovascular system, others attribute this role to TMA, even hypothesizing that TMAO has a protective effect from damage induced by TMA [27]. From the molecular docking analysis, it emerges that, even if TMAO and TMA have a calculated comparable free binding energy with HSA (Table S1), they do not have the same binding affinity. In fact, TMAO binds only to externally located binding sites, i.e., in proximity of the HSA molecular surface limit. This corresponds to labile and not durable binding interactions, that are instead observed for TMA, whose poses are buried inside the HSA helices and fixed via direct interaction with a huge number of residues, such as disulphide linkages. These in silico findings are coherent with previous observations suggesting that incubation with TMA but not TMAO resulted in the change of conformation of the protein structure of albumin [28]. However, in the present study, the tryptophan emission peak was significantly (\(p\) = 0.010) lower when albumin was incubated with both TMA+TMAO starting from low concentrations, which mimics physiological conditions [15]; this suggests a synergistic cooperative interaction of TMA and TMAO with HSA. The decrease in Trp fluorescence emission intensity may be associated with the changes in the microenvironment around Trp 214 following the interaction with TMA and TMAO. It can be hypothesized that the weak interactions with TMA and TMAO do not promote any HSA oxidation since no significant differences were observed in carbonyl groups formation. According to this outcome, under our experimental conditions, a stabilizing effect of TMA+TMAO on HSA could be hypothesized.