Potential of triacylglycerol pro ﬁ les in latent ﬁ ngerprints to reveal individual diet, exercise, or health information for forensic evidence †

Triacylglycerols (TGs) are a primary component of human skin oils and are therefore a major constituent present in latent ﬁ ngerprint residue. We hypothesize that di ﬀ erences in the TG pro ﬁ le of the latent ﬁ ngerprint, such as the relative level of saturation of the fatty acyl chains, may link back to the overall health of the subject, speci ﬁ cally correlated to diabetes. A small scale proof-of-concept study was performed to test this hypothesis. The biggest di ﬀ erences observed were in the relative amounts of completely saturated TGs compared to TGs with one or more double bonds; however, hierarchical clustering could not di ﬀ erentiate diabetic and non-diabetic participants. The role of other factors such as diet and exercise was explored from subsets of the non-diabetic participants. Vegetarian participants had higher relative levels of saturated TGs compared to those without diet restrictions; however, people on a low carbohydrate or ketogenic diet were not distinguishable from the control population. In terms of exercise, male participants who were very active had lower levels of saturated TGs compared to males who did not exercise at all. In contrast, exercise did not have much of an e ﬀ ect on the TG pro ﬁ le of female participants. While the outcome of this study is only tentative due to the lack of statistical power, it suggests the potential of TG pro ﬁ les to be linked to health information, diet and exercise.


Introduction
Fingerprints are one of the most common means of personal identication and have been used in the criminal justice system for more than a century. However, there are several limitations in the way latent ngerprints are currently analyzed. The biggest limitation with this workow is that ngerprints are useful evidence only if a match for the obtained ngerprint is found in the database. If the ngerprint cannot be connected back to a specic individual, it is not useful for evidence. Recent research is looking to increase the evidentiary value of such ngerprints by obtaining additional information from the chemical prole of the ngerprint. [1][2][3][4] Latent ngerprints consist of chemicals that are naturally present in sweat or those that are excreted through pores in the nger. These chemicals are known as endogenous compounds and include fatty acids, amino acids, and triacylglycerols (TGs). Fingermarks may also contain chemicals with which the nger has come into contact, oen called exogenous compounds. These can include anything from consumer products to illicit drugs and explosives. Previous research efforts have studied endogenous compounds in latent ngerprints to determine traits that correspond to the donor. 5 For example, amino acids are known to be useful for determining the sex of the subject 6 and lipid proles have been used to narrow down the age of the subject. 7 Research has also shown that exogenous compounds in ngerprints can be used to connect an individual to a certain lifestyle or illicit activity. [8][9][10][11] Mass spectrometry imaging (MSI) provides the advantage of simultaneous collection of both chemical and spatial information, making it popular for the analysis of latent ngerprints because an image that could be used for a database search is generated while additional chemical information is being obtained. As a surface technique, MSI requires very little sample preparation. This is in contrast to gas chromatography (GC) and liquid chromatography (LC)-MS techniques, which both require lengthy extractions. Additionally, GC-MS and LC-MS oen require the extraction of 3-5 ngerprints to get good signal for endogenous compounds, 12 whereas MSI only requires one. Moreover, the imaging capability of MSI allows for the differentiation of ngerprint compounds from surface contaminations. Desorption electrospray ionization (DESI) 13 and secondary ion mass spectrometry (SIMS) 14 have both been utilized for imaging of latent ngerprints; however, matrix assisted laser desorption/ionization (MALDI) has become the most popular technique for this application because it is robust, versatile, and minimally destructive. 1,15 Fingerprints have almost no apparent damage aer MALDI-MSI analysis and could be used as evidence in court proceedings or for additional analysis. Furthermore, MALDI-MSI is useful for forensic analysis of latent ngerprints because it has proven to be compatible with current forensic development techniques, making integration into labs more likely. 16,17 TGs are one of the most common classes of compounds found in latent ngerprints and consist of a glycerol backbone and three fatty acid chains of various lengths and double bonds. Attempts have been made to use TGs to distinguish particular traits such as sexes, 18-20 races, 21 ages, 7 and diets; 20 however, each case was found to be inconclusive, not statistically signicant, or only tentative due to a relatively small sample size. As TGs are involved in many health conditions, 22 we hypothesize the TG proles in latent ngerprints could potentially identify health information of individuals, specically how they relate to metabolic diseases such as diabetes. Previous research has shown free fatty acid composition, such as chain length and level of unsaturation, effect the release of insulin, 23,24 suggesting a potential correlation between fatty acyl chains on TGs and diabetes. This could be useful in the forensic eld because the ability to determine if an unknown suspect is diabetic would drastically narrow the suspect pool. In addition, this could also have potential applications in medical diagnostics. Analyzing a latent ngerprint is extremely non-invasive, making it a potentially very useful diagnostic tool. In this work, a small scale study is conducted to test the hypothesis that the TG prole in latent ngerprints is a feasible way to determine health information about an individual, specically in relation to diabetes.

Sample collection and preparation
In order to determine the variability in the TG prole of an individual, a single donor gave a ngerprint at ve different times over the course of several months. These ngerprints were prepared as described below and were run separately on the mass spectrometer, taking both individual and instrument variability into account.
Volunteers were recruited among the students, staff, and faculty of Iowa State University for ngerprint donation. All methods involving the research on human subjects was approved by our institutional review board. Participants were asked to briey rub a nger against their forehead before lightly placing it on a glass slide. Groomed ngerprints were used to ensure good TG signals from all participants. 79 total participants were recruited and they varied in sex, race, age, and lifestyle. There were 36 male and 43 female participants and 16 total who had previously been diagnosed with diabetes (no discrimination between type 1 and type 2 diabetes). Fingerprints were collected 4-8 at a time so they would all t in the sample plate at the same time and were stored in a desiccator until analysis (desiccator storage time did not exceed four hours).
Once the ngerprints were collected, they were sprayed with a sodium acetate (Alfa Aesar, Ward Hill, MA) solution and sputter coated with a gold target (Ted Pella Inc. Redding, CA). The sodium acetate (10 mM in methanol) was applied using a TM Sprayer (HTX Technologies LLC, Chapel Hill, NC). The TM Sprayer method included 8 passes at 30 C with a ow rate of 0.03 mL min À1 and a linear velocity of 1200 mm min À1 . The gold sputter coating was done at 40 mA for 10 seconds ($2.8 nm thickness) 25 on a 108 Auto Sputter Coater (Ted Pella Inc. Redding, CA). The combination of spraying sodium acetate and sputter coating with gold was modied from a previously reported method. 26 Instrumentation Data was exclusively collected on a MALDI-linear ion trap (LIT)-Orbitrap mass spectrometer (MALDI-LTQ-Orbitrap Discovery; Thermo Finnigan, San Jose, CA). The mass spectrometer includes a modication to incorporate an external frequency tripled 355 nm Nd:YAG laser (UVFQ; Elforlight, Daventry, UK). Mass spectra were collected in positive ion mode using the Orbitrap mass analyzer from m/z 500-1000 with 10 laser shots per spectrum and a raster step of 100 microns. Data analysis was done using Xcaliber and ImageQuest (ThermoFisher Scientic).

Hierarchical clustering
Mass spectra were averaged over the entire region of the ngerprint that was imaged. Hierarchical clustering was done using the online statistical soware MetaboAnalyst. 27 The data submitted to MetaboAnalyst was a list of the normalized TG signal for the 20 most abundant TGs observed in the latent ngerprints of each participant. The TGs were normalized to the most abundant TG in the spectrum.

Day-to-day variation in an individual
First, an experiment was performed to determine the variability for a single individual and the best method to minimize day-today variation. Previous research has shown that there is a fair amount of variability in the chemical prole of a ngerprint made by the same donor on different days. 28 Weyermann et al. showed that cholesterol and squalene levels measured by GC-MS were different not only from day to day, but also in different measurements on the same day. Similarly, we experienced differences in the absolute signal of TG species in our experiment. ESI Fig. 1 † shows variation in absolute signal of the most abundant TG over three separate 24 hour time periods. In order to account for some of this variability, TG species were normalized to the most abundant TG in each ngerprint. ESI Fig. 2 † shows three normalized TG proles that were extracted from these trials and look similar regardless of the time of day. The normalized ratios proved to be relatively consistent with relative errors of less than 10% for each TG. Fig. 1 shows the average TG prole for a single donor on ve separate days (and at different times of day) over the course of several months. The standard deviations were also for the most part less than 10%, with a few exceptions for TGs with low absolute signal. Similar results were obtained regardless of individuals or instrument conditions. These results indicate that normalized TG proles are a reliable way to compare between individuals. Thus, our subsequent study was focused on comparing relative TG patterns (i.e., fatty acyl chain length and unsaturation) depending on health information.

Impact of diabetes on TG prole
A small scale study was performed with the intent to determine if TG proles could be used as a means to distinguish between diabetic and non-diabetic individuals. In this study, ngerprints were collected from 79 total individuals (36 males and 43 females). This number includes 16 people who have previously been diagnosed with diabetes and 63 healthy individuals. Six representative MS images of different TGs detected in the ngerprints of three participants are shown in Fig. 2. Each row has an odd and an even chain length TG from the same individual, both of which are localized to the ngerprint ridges.
While the color (representing the signal intensity) may be slightly different between images, each TG is localized to the ngerprint ridges regardless of chain length or level of unsaturation. It has been well known that human sebaceous glands produce unusual surface lipids containing odd chain fatty acyl chains. 29 A visualization of the twenty most abundant TG proles is made as a heat map in Fig. 3, with hierarchical clustering analysis using MetaboAnalyst (https://www.metaboanalyst.ca/). It clusters participants with similar TG proles together in the same part of the heat map. The columns in the heat map represent each individual participant and the rows represent each specic TG species. The color of each square indicates the relative intensity of the given TG for each individual, with red meaning there is a higher relative intensity for that peak than blue.
The heat-map is broken up into three main clusters of participants. No particular TG species stands out more than the others; instead it seems the abundance of overall unsaturated TG species plays a key role in this clustering. Group I consists of individuals with overall high levels of unsaturated TG species. In contrast, Group III has mostly low levels of unsaturated TGs. Group II seems to have relatively good balance of high and low unsaturated TGs. As Group II is the largest, it can be split up into two subgroups: Group IIA and Group IIB. The best distinguishing feature between the two subgroups is that Group IIA has higher relative abundance of saturated TGs than Group IIB. Most diabetic participants (14 out of 16) are present in Group II. Within Group II, the diabetics are pretty evenly split between Groups IIA and IIB, although there are more type 2 diabetes participants (3 out of 4) in Group IIA. Overall, there is no clear distinction between diabetic participants and healthy individuals.
There is a fair amount of variation even among the healthy controls. Some healthy individuals were clustered in Group I with high unsaturated TGs and low saturated TGs, while some healthy individuals show the opposite trend in Group III. There are some general trends suggesting diabetes patients have more abundant saturated TGs, especially for type 2 diabetes (i.e.,  Group IIA); however, this effect is obscured by other factors in the current small scale study. For example, most of the participants in Group III with elevated saturated TGs were participants who do not exercise at all or rarely exercise. For a better understanding of other factors, the data set for healthy individuals was carefully examined to nd any correlations between diet or exercise and relative TG saturation levels. The results are discussed in the following sections.

Impact of exercise on TG prole
Aer noticing the similarities between the participants in Group III, the effect of exercise on TG proles was explored. Out of 63 total healthy individuals from the previous section, ngerprint data from 8 male and 8 female participants were separated out for further data analysis. These individuals were chosen because their surveys indicated they had a very clear exercise routine (either they exercised regularly or not at all) while also having a similar diet. These 16 participants all had an average diet with three meals per day and no diet restrictions. The male participants include 4 that exercise every day and 4 that do not exercise, whereas the female participants include 5 that exercise on a regular basis, and 3 that do not exercise. The most apparent difference between the two groups is the highly abundant saturated fatty acyl chains (i.e., TG x:0) among the non-active males compared to the active males as shown in ESI The result is summarized in Fig. 4 for the three most abundant saturated TGs normalized to the most abundant TG in the series with the same number of carbons. Other normalization strategies were also tested and similar results were achieved (data not shown). For most participants, the monounsaturated TG was the most abundant in the series (i.e., TG x:1), although some people had the doubly unsaturated or saturated TG as the most abundant. The three most abundant saturated TGs were chosen in an effort to achieve a ratio that best represents the individual. The error bars on these graphs are relatively large because they combine the TG proles for different individuals. The results indicate that exercise has a larger effect on the TG proles of males than females. The males who exercise daily have signicantly lower levels of saturated TGs compared to non-active males (44%, 46%, and 36% of the relative abundance for non-active males for TG 46:0, TG 47:0, and TG 48:0, respectively), with pvalues less than 0.05 or 0.01. Active females also have lower levels of saturated TGs compared to non-active females, but the difference is relatively minor (78%, 80%, and 72% for TG 46:0, TG 47:0, and TG 48:0, respectively) and not statistically signicant (p > 0.3). TG 48:0 seems to have the biggest effect on both male and female participants with the active participants having relative abundances 36% and 72% of the non-active participants respectively. Overall, this data suggests that routine exercise results in lowering the saturated to unsaturated TG ratios on ngerprints, especially in males. Similar effects have been previously reported in literature for the effect of exercise on TGs in serum. 30,31 Impact of diet on TG prole Next, the effect of diets on relative TG patterns was studied. TG proles from twelve healthy participants were extracted from the original diabetes data set that have a clear diet pattern. This group of twelve includes 5 vegetarians, 3 people with low carbohydrate/ketogenic diets, and 4 people that reported no diet restrictions. Each of these participants had similar exercise routines, exercising regularly several times per week. This subset data for diet includes 4 males and 7 females, but there are no apparent differences between male and female participants that are a part of the same diet group. The three most abundant saturated TG species were compared and are shown in Fig. 5. There is no signicant difference in the relative abundance of saturated TGs between the low carbohydrate and unrestricted diets (p values of 0.06-0.2). However, the vegetarians had signicantly higher levels of saturated TGs on average than either of the other two diets (p < 0.05). Unlike the effect of exercise in the previous section, the relative differences in the three most abundant saturated TGs are similar between the vegetarian and control groups (1.9, 1.7, and 1.9 times higher in the vegetarian group than the control for TG 46:0, TG 47:0, and TG 48:0, respectively). This suggests that vegetarians could have higher levels of saturated TGs compared to people without any diet restrictions.

Conclusions
The presented data from this small proof of concept experiment shows that the relative abundance of TGs in latent ngerprints may be impacted by lifestyle choices such as diet and exercise or health conditions such as diabetes. While the current small scale study was inconclusive in regards to diabetes, the elevated relative abundance of completely saturated TGs is partially correlated with diabetes, particularly for type 2 diabetic participants. This correlation is complicated by other factors, as the data from small subsets of healthy participants suggests. Male participants in particular had much lower levels of saturated TGs when they had an active lifestyle and exercised every day compared to inactive individuals. On the other hand, the TG proles of female participants were not signicantly impacted. Individuals with a vegetarian diet also had higher than average saturated TGs compared to people that had no restrictions to their diet. Despite the fact that the current nding is only tentative, this study leads to an important discovery that there might be a potential correlation between TG proles and lifestyle or health status. To further study the effect of diabetes on TG proles, a large scale study is currently being planned in the coming years with a much larger population of type 2 diabetics that carefully controls for diet and exercise. If this can be successfully accomplished, it could lead to signicant applications not only in forensics, but also diagnostics as a quick and non-invasive procedure.

Conflicts of interest
The authors declare no conicts of interest. The research was conducted in the absence of any nancial or commercial  This journal is © The Royal Society of Chemistry 2020 Anal. Methods

Paper Analytical Methods
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