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Bloodstain age estimation involves measuring time-dependent changes in the levels of biomolecules in bloodstains. Although several studies have identified bloodstain metabolites as markers for estimating bloodstain age, none have considered sex, age-related metabolomic differences, or long-time bloodstain age. Therefore, we aimed to identify metabolite markers for estimating the age of bloodstains at weekly intervals within 28 days and validate them through multiple reaction monitoring. Adenosine 5′-monophosphate, choline, and pyroglutamic acid were selected as markers. Seven metabolites were validated, including five previously reported metabolites, ergothioneine, hypoxanthine, L -isoleucine, L -tryptophan, and pyroglutamic acid. Choline and hypoxanthine can be used to differentiate bloodstains between days 0 and 14 after deposition at weekly intervals, whereas L -isoleucine and L -tryptophan can help distinguish bloodstains between 7 days before and 14 days after deposition. Evaluation of the changes in metabolite levels according to sex and age revealed that the average levels of all seven metabolites were higher in women on day 0. Moreover, the level of ergothioneine was significantly higher in elderly individuals than in young individuals at all time points. In this study, we confirmed the potential effectiveness of metabolites in bloodstains as forensic markers and provided a new perspective on metabolomic approaches linked to forensic science. [ABSTRACT FROM AUTHOR]

Ergothioneine, which is a naturally occurring metabolite, generally accumulates in tissues and cells subjected to oxidative stress, owing to its structural stability at physiological pH; therefore, it has been attracting attention in various biomedical fields. Ergothioneine has also been suggested as a potential forensic marker, but its applicability has not yet been quantitatively validated. In this study, quantitative analysis of ergothioneine in bloodstains was conducted to estimate the age of bloodstains and that of bloodstain donors. Blood from youth and elderly participants was used to generate bloodstains. After extracting metabolites from the bloodstains under prevalent age conditions, ergothioneine levels were quantified by mass spectrometry via multiple reaction monitoring. The concentration of ergothioneine in day 0 bloodstains (fresh blood), was significantly higher in the elderly group than in the youth group, but it did not differ by sex. Statistically significant differences were observed between the samples from the two age groups on days 0, 5 and 7, and on days 2 and 3 compared with day 0. The findings suggest that ergothioneine can be used to estimate the age of bloodstains and of the donor; it could be useful as a potential marker in reconstructing crime scenes. [ABSTRACT FROM AUTHOR]

Bloodstains are frequently encountered at crime scenes and they provide important evidence about the incident, such as information about the victim or suspect and the time of death or other events. Efforts have been made to identify the age of the bloodstain's donor through genomic approaches, but there are some limitations, such as the availability of databases and the quality dependence of DNA. There is a need for the development of a tool that can obtain information at once from a small blood sample. The aim of this study is to identify bloodstain metabolite candidates that can be used to determine donor age. We prepared bloodstain samples and analyzed metabolites using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eighteen molecular features (MFs) were selected as candidates using volcano plots and multivariate analysis. Based on the MS/MS spectrum of the MFs, the following nine metabolites were identified from the METaboliteLINk database: Δ2-cis eicosenoic acid, ergothioneine, adenosine 5′-monophosphate, benzaldehyde, phenacylamine, myristic acid ethyl ester, p-coumaric acid, niacinamide, and N-arachidonoyl-l-alanine. These nine age markers at high or low abundances could be used to estimate the age of a bloodstain's donor. This study was the first to develop metabolite age markers that can be used to analyze crime scene bloodstains. [ABSTRACT FROM AUTHOR]

Bloodstains found at crime scenes contain immense information about the crime; thus, studies involving analysis of small molecules in bloodstains have been conducted. However, most of these studies have not accounted for the difference in the results of small molecule analysis due to the surface of bloodstains. To evaluate the "surface effect," we prepared bloodstains on seven surfaces, including both absorbent and non-absorbent surfaces, and performed global small molecule analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). We used three indicators: (1) count recovery rate (%) of molecular features (MFs), (2) the number of MFs extracted from the surface without bloodstains, and (3) difference in abundance recovery rate (%) of MFs, to determine the ranking of the seven surfaces in the order of their similarity with blood. We also confirmed the correlation between each surface and blood through multivariate analysis. We found that the non-absorbent surfaces ranked better than the absorbent surfaces; wooden flooring was ranked as the most efficient surface, followed by stainless, vinyl flooring, glass, tile, filter paper, and mixed cotton. This study will help in the selection of the most efficient surface for collection of bloodstains for small molecule analysis from a crime scene. This is the first study to identify the effects of surface on extraction of global small molecules from bloodstains; it will help forensic scientists in obtaining more accurate information from small molecules present in the bloodstains collected at the field. [ABSTRACT FROM AUTHOR]

Establishing a correlation between environmental variables and chemical change can significantly improve the quality of research in multiple fields. Among various environmental variables, temperature and humidity are closely related to the rate of chemical reactions. This study aimed to confirm changes in metabolite markers that were previously discovered in other temperature and humidity environment conditions and to confirm the possibility that they could act as markers. After blood collection from the subjects and bloodstain preparation, the quantitative values of the bloodstain metabolites were confirmed (when the age of the bloodstain was within a month) under eight environmental conditions (4 °C/30%, 4 °C/60%, 25 °C/30%, 25 °C/60%, 25 °C/90%, 40 °C/30%, 40 °C/60%, and 40 °C/90%). Age-of-bloodstain estimation models were constructed to confirm the applicability of bloodstain metabolites as markers for bloodstain age in various environments. The average concentration of metabolite markers exhibited a decreasing trend with the age of the bloodstain, which transformed into an increasing trend from day 7 onwards. In terms of temperature and humidity, 25 °C and 90%, respectively, showed the most dissimilar metabolite change pattern compared to other conditions. The age-of-bloodstain estimation models developed here have an R-square value of up to 0.92 for each condition and an R-square value of 0.71 when all environmental conditions were combined. The findings herein highlight the immense potential of blood metabolites for field application, confirming the possibility of predicting metabolite changes from the rates of their chemical reactions and validating the importance of metabolites as age-of-bloodstain markers under various environmental conditions. • Uncontrolled chemical changes in an ex-vivo environment are poorly understood. • Bloodstains in environmental conditions were examined using metabolomic approach. • Bloodstain metabolites showed the greatest change at 25 °C and 60% relative humidity. • Metabolite marker concentrations showed an increasing trend after 7 days. • Prediction models allowed for estimating the age of bloodstain using metabolites. [ABSTRACT FROM AUTHOR]