## 4 Discussion  
Chemical and physical properties of biological traces are directly affected by oxidative modifications of proteins and lipids. However, the influence of protein and lipid oxidation on the ageing of body fluids ex vivo is poorly understood. Currently, there is an increasing interest among forensic scientists to acquire knowledge about the ageing of body fluids for the development of age estimation methods [32]. As such, in the present pilot study, different parameters to assess protein and lipid oxidation were determined for the first time using fluorescence spectroscopy, ELISA, and SPR. Fluorescence spectroscopy is commonly used in biomedical science to quantify protein oxidation. [PERSON] et al. used fluorescence spectroscopy to evaluate the oxidation of bovine serum albumin (BSA) and dairy proteins [16]. Protein oxidation was determined by measuring the loss of the natural tryptophan fluorescence and the gain of protein carbonyls [16]. Similar kinetics were observed by [PERSON] and [PERSON] in ageing of fingermarks and semen, respectively [11; 12]. However, since non-contact with the body fluid is necessary to allow the use of this technique in forensic practice, the researchers measured the fluorescence with a fiber-based accessory. To eliminate effects of height differences of the fiber between the time intervals, the ratio was used as measure of protein oxidation. The maximum fluorescence intensities of protein fluorescence over time were measured between 320-340 nm for semen and saliva and between 340-370 nm for urine. Figure 6: Overview of oxidation parameters in saliva during ageing. (**A**) Median protein/FOX ratio over time, with whiskers indicating the range from the smallest to the largest ratio. (**B**) Mean protein carbonyl concentrations (nmol/mg) increases over time with error bars representing standard deviation. Note that The median protein/FOX ratio decreased over time, while the protein carbonyl concentrations (nmol/mg) increased. The second peak, which represents the fluorescent oxidation products, was observed between 400-450 nm for semen and urine samples. For saliva, low fluorescence intensities could be observed at 430 nm. Low fluorescence of the fluorescence oxidation products was expected due to the low presence of PUFAs in saliva [33]. Saliva was the only body fluid for which a significant difference in the fluorescence ratios could be observed over time. The decrease in fluorescence ratios is in agreement with previous findings reported in the literature [31]. Carbonyl compounds are products of protein oxidation that are involved in cross-linking of damaged proteins via Schiff base formation [34]. Schiff bases can be formed when aldehydes, such as MDA and HNE, react with amino groups from the side chain of proteins. They can be detected around 450 nm upon excitation at 350 nm, which corresponds with the emission spectrum of our unknown fluorescent products. Protein carbonyls were detected in all three body fluids; however, only low concentrations (varying from 0.14 \(\pm\) 0.06 to 0.892 \(\pm\) 0.02 nmol/mg) of protein carbonyls could be detected in urine and therefore high concentrations of MDA, HNE, and AGE were not expected as these are involved in the formation of protein carbonyls. This is in agreement with our study results (Figure 3B), as we observed that these markers could only be detected in a few samples. We hypothesize that, as the fluorescence ratio decreases, carbonyl protein concentration increases. The protein carbonyl concentrations increased over time for semen and saliva, which indicates that protein carbonyl contributes to the emission spectrum of fluorescence oxidation products but may not be a key player. Only for saliva could a statistically significant difference be determined, but the use of a larger sample sizes may improve the statistical significance of these results. A significant difference could be used as a measure to distinguish between fresh and aged samples.