key: cord-0049978-bxwefat6
authors: Hyde, Jacob; Runyon, J. Ray
title: LCMS Measurement of Steroid Biomarkers Collected from Palmar Sweat.
date: 2020-09-09
journal: ChemRxiv
DOI: 10.26434/chemrxiv.12931769
sha: 83c04cfc623656021cc45468297837cbeb6d1607
doc_id: 49978
cord_uid: bxwefat6

Human eccrine sweat contains numerous biomarkers which can provide information on health,performance, and aging. Non-invasive collection and measurement of biomarkers has becomeespecially important in recent times given viral outbreaks like SARS-CoV-2. In the current studywe describe a method of sweat collection from palmar surfaces in participants via surface captureusing glass beads and the resulting analysis of biomarkers from very low volumes of sweat usingliquid chromatography mass spectrometry with selected ion monitoring. Study participantsunderwent a cognitive and physical stress task with easy and hard conditions with sweat beingcollected after each task. Resulting analysis found a signal for 22 steroid biomarkers and wereport detailed information on selected biomarkers, given their applicability to timely real-worldexemplars, including cortisol, dehydroepiandrosterone, allopregnanolone, estrone, aldosterone,and 20a/b-dihydrocortisone.

Human eccrine sweat contains numerous biomarkers which can provide information on health, 27 performance, and aging. Non-invasive collection and measurement of biomarkers has become 28 especially important in recent times given viral outbreaks like SARS-CoV-2. In the current study 29

we describe a method of sweat collection from palmar surfaces in participants via surface capture 30 using glass beads and the resulting analysis of biomarkers from very low volumes of sweat using 31 liquid chromatography mass spectrometry with selected ion monitoring. Study participants 32 underwent a cognitive and physical stress task with easy and hard conditions with sweat being 33 collected after each task. Resulting analysis found a signal for 22 steroid biomarkers and we 34 report detailed information on selected biomarkers, given their applicability to timely real-world 35 exemplars, including cortisol, dehydroepiandrosterone, allopregnanolone, estrone, aldosterone, 36 and 20a/b-dihydrocortisone. Sweat is a biofluid rich in biomarkers that can provide information about human cognitive 50 performance, health, disease state(s), nutrition and environmental impacts. Steroids and other 51 immune biomarkers have been detected in eccrine sweat (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) . Non-invasive measurement tools 52 of biomarkers are needed and have become especially salient given recent viral outbreaks like 53 SARS-CoV-2, where in-person blood collection is not feasible or unadvisable. The monitoring of 54 some biomarkers may help to monitor human performance in industrial and occupational 55 settings, may help to predict and track acquired disease processes like obesity, and may help to 56 determine how psychiatric sequalae are experienced and expressed. 57 58 Steroids of interest for the monitoring and enhancement of human performance and health are 59 many and include cortisol, dehydroepiandrosterone (DHEA), allopregnanolone, estrone and 60 aldosterone. Cortisol is secreted in response to stress, suppressing the hypothalamic-pituitary-61 adrenocortical (HPA) axis and negatively effecting health and cognition (13). DHEA is 62 implicated in many physiological processes with impacts on disease resistance and immune 63 function (14) . DHEA and cortisol are the most common products of the stress response from the 64 endocrine system, mediating short and long-term stress responses via the HPA-axis; DHEA can 65 be converted into dehydroepiandrosterone sulfate (DHEA-S) and has been shown to antagonize 66 the effects of cortisol (15). In humans, basal DHEA levels can be altered by exposure to 67 traumatic events like military combat and lower cortisol/DHEA ratios have been found in 68 patients with post-traumatic stress disorder (PTSD) (16). Positive correlations have also been 69 found between current PTSD symptoms and the ratio of DHEA to allopregnanolone (17). 70

Allopregnanolone is a metabolite of progesterone and plays a role in neuronal excitability at the 71 synaptic and extrasynaptic ƴ-aminobutyric acid (GABAA) receptor (18); GABAergic 72 neurotransmission has been shown to be impaired in subjects with PTSD (19). Furthermore, 73 allopregnanolone is a key therapeutic target for research and development of neurodegenerative 74 and age-related diseases (20). Estrone is one of the three most common estrogens found in 75 humans. In men, estrone has been shown to increase as BMI rises (21) and may be a sensitive 76 marker of acquired Type-II diabetes risk (22). Aldosterone, through the Renin-Angiotensin-77

Aldosterone System (RAAS) has health implications for the vascular, renal and cardiovascular 78 systems, which is of heightened interest due to the SARS-CoV-2 virus and resulting COVID-19 79 disease (23). In this study, participants followed prompts on the screen from a program designed in 127 MATLAB. A neurocognitive task (N-back 1&2 digits) was presented with two difficulty 128 conditions (easy/hard). Participants then engaged with a physical task (finger/keyboard tapping) 129 with two difficulty conditions (easy/hard) which were randomized within task for each 130 participant. 131 132 A batch of 4mm glass beads made from borosilicate were baked at 475 ºC for 4 hours to remove 133 organic contaminates, allowed to return to room temperature, divided into 35 bead aliquots and 134 stored at room temperature in falcon tubes until use. Participants had these glass beads poured 135 from the tubes into their hands and then instructed to roll them between the palms of their hands 136 for 60 seconds. After collection of these samples, the beads were returned to the containers, and 137 then were stored at 4 ºC until biomarker analysis. Sample collection was performed at the 138 beginning of the study session, after completion of N-back cognitive tasks, and after completion 139 of physical tasks. 140 Restek Raptor Biphenyl column (200 mm x 2.1 mm; 2.7 um bead) held at 30C and a water/ACN 148 (0.1% formic acid) binary solvent system. The solvent flow rate was set to 0.5 mL/min with a 149 gradient of ACN from 10% -100% over 3 minutes followed by a column washing step for 1-150 minute at 100% ACN and a 3-minute column equilibrium step at 10% ACN before the next 151 sample injection. Mass spectrometer settings used for detection of steroid biomarkers were as 152 follows: 3kV in positive ion mode, 0.4 resolution, 20usec dwell time, 300 ºC vaporizer 153 temperature and ion transfer tube temperature, 50abs N2 sheath gas, 25Arb N2 aux gas. 

The results shown below in Figure 1 summarize the different steroid molecules that were found 160 in palmar sweat utilizing an adapted LCMS methodology from (11). 161 Figure 1 . LCMS-ESI-SIM chromatograms for steroid biomarkers found in palmar sweat. 163

Progestogens (yellow), corticosteroids (green), androgens (blue), estrogens (pink), cholesterol 164 (white). 165 166 and high rates of obesity (BMI >30kg/m 2 ) at 44% (31). While still relatively young, these 236 complex conditions are unlikely to resolve as this cohort continues to age, further complicating 237 typical age-related physical issues and normal age-related cognitive decline. As previously 238 noted, biomarkers like cortisol, DHEA, allopregnanolone, estrone and aldosterone are all 239 implicated and play a role in the moderation of these conditions. Further, monitoring the status of 240 steroids and immune biomarkers of servicemembers during their active-duty service may provide 241 useable and actionable information necessary to enhance overall performance and potentially 242 decrease the likelihood of negative outcomes after service. In veterans and the general 243 population, biomarker monitoring may help to predict and determine the onset of disease, the 244 course of disease processes, and may influence precision medicine strategies. 245

We have now shown that using non-invasive methods of eccrine sweat collection in very low 247 volumes of sweat is possible via surface capture using glass beads. Multiple biomarkers can be 

Neural control of sweat secretion: a 261 review

Physiology of sweat gland function: The roles of sweating and 263 sweat composition in human health

Development of a sensitive 267 microarray immunoassay for the quantitative analysis of neuropeptide

Cortisol in Human Eccrine Sweat by Liquid Chromatography -Tandem Mass 271

The 274 microfluidics of the eccrine sweat gland, including biomarker partitioning, 275 transport, and biosensing implications

Molecular cartography of the human skin surface in 3D

Kamin HS, Kertes DA. Cortisol and DHEA in development and 312 psychopathology

DHEA-S levels in posttraumatic stress disorder: A meta-analytic review

Decreased Cerebrospinal Fluid Allopregnanolone Levels in Women 320 with Posttraumatic Stress Disorder

Allopregnanolone: From molecular 324 pathophysiology to therapeutics. A historical perspective

Neurosteroid Levels in the Orbital Frontal Cortex of Subjects With PTSD 329 and Controls: A Preliminary Report. Chronic Stress

Allopregnanolone as a Therapeutic

Regenerate the Degenerated Brain

Frontiers in Gynecological Endocrinology

Impact of age, body weight and metabolic risk factors on steroid 340 reference intervals in men

Circulating Estrone Levels Are Associated Prospectively With Diabetes Risk 345 in Men of the Framingham Heart Study. Diabetes Care

Understanding the Renin-Angiotensin

Axis: A Comprehensive Review

Ultra Performance Liquid Chromatography-Tandem Mass 355

Spectrometry Method for Profiling of Steroid Metabolome in Human Tissue

Accessing analytes in biofluids for peripheral biochemical monitoring

Working Up a Good Sweat -The 363

Challenges of Standardising Sweat Collection for Metabolomics Analysis

Timchenko 367 MA. Analyzing and Mapping Sweat Metabolomics by High-Resolution

PLoS One

Personal Information from Latent Fingerprints Using 371

Desorption Electrospray Ionization Mass Spectrometry and Machine 372

Thermographic imaging for detection of changes in autonomic 376 nervous system activity and response to varying stress levels through changes 377 in sweat pore activation

The 379 prevalence of posttraumatic stress disorder in Operation Enduring 380

Freedom/Operation Iraqi Freedom (OEF/OIF) Veterans: a meta-analysis

Risk Factors of Obesity in 383

Veterans of Recent Conflicts: Need for Diabetes Prevention