## 4 Discussion
Furthermore, FTIRI images of proteins showed a spot arrangement, similar in both callosal regions (genu and splenium), as also suggested by the PRT/TBM ratio, which did not display statistically significant differences among groups. Moreover, the peak-fitting procedure highlighted statistically significant higher amounts of the saturated (\(A_{\text{CH2}}/A_{\text{CH3}}\) band area ratio) and unsaturated (\(A_{\text{CH}}/A_{\text{CH3}}\) band area ratio) lipids found in the splenium than the to genu, and in younger than older specimens. This biochemical variation of lipids was best described by the CH/CH3 ratio, which indicated the content of unsaturated lipids, and by the CH2/CH3 ratio, which was related to the length of the alkyl chains; it is known that the oxidative stress-related intracellular free radicals trigger lipid peroxidation, mainly affecting unsaturated ones, leading to their rapid degradation and, hence, clearance [44; 49; 50]  
The relationship among lipid and protein components represents an index of the stability and integrity of the myelin membrane. To evaluate this aspect, the LIP/PRT ratio was calculated. In the old group, lower values were measured than in the young group in both the analyzed regions, indicating a reduction of the lipidic component due to the phenomenon of oxidation; moreover, both in younger and older samples, the splenium displayed a higher value of the LIP/PRT band area ratio than the genu. Furthermore, the shorter length of the chains, caused by fragmentation, lipid degradation, and protein denaturation, caused an increase in membrane fluidity, with a loss of stability and integrity, leading to demyelination [51]. Demyelination has been observed to begin in middle age and continues slowly over the years, following a rostrocaudal trajectory [26]. Similar amounts of proteins were found in all the experimental groups (PRT/TBM band area ratio), regardless of the age or region. Nevertheless, some interesting differences were observed about their secondary structure. In general, higher amounts of random coil structures (RANDOM and FOLDED/UNFOLDED band area ratios) were found in older specimens. These results agreed with several studies, which specified the role of oxidative stress on the biochemical composition of macromolecules [35; 51; 52]. It is known that oxidative stress, caused by reactive oxygen species (ROS) accumulation, is one of the mechanisms responsible for aging and is capable of causing changes in the secondary structure of proteins, including a partial conversion of \(\alpha\)-helices in \(\beta\)-sheets, and a fragmentation and denaturation of the amino acid chains with consequent loss of functionality [35; 51; 52]. Although the present results showed an increase in random disordered structures with advancing age, the same transformation was not observed for the \(\beta\)-sheets, which appeared to decrease in the older compared to the younger samples. This research represents the first FTIR investigation on the human CC; therefore, results from other investigations are not available. A recent study with a similar purpose has been carried out on rats to study the biochemical changes provoked by traumatic axonal injury (TAI) [34; 35] and traumatic brain injury (TBI) [51] in an animal model, and the age-related changes in the WM and CC in the rat brain [36]. In these studies, in the injured groups, FTIR revealed changes within the rat CC related to protein conformation (increased \(\beta\)-sheet and \(\beta\)-turn contents together with a decrease in \(\alpha\)-helix content), and a significant decrease in the lipid/protein ratio. Moreover, FTIR was also able to discriminate normal from injured WM at different time points after TBI by examining these infrared spectral parameters [34; 35], suggesting that this kind of analysis could also be useful in neurological and forensic fields. A more recent study applied FTIR spectroscopy to investigate biochemical changes after TBI in cortex and CC of rats [51].