Relationship between the concentration of ergothioneine in plasma and the likelihood of developing pre-eclampsia

Ergothioneine, an antioxidant nutraceutical mainly at present derived from the dietary 14 intake of mushrooms, has been suggested as a preventive for pre-eclampsia. We analysed early 15 pregnancy samples for a cohort of 432 first time mothers as part of the Screening for En dpoints in 16 Pregnancy (SCOPE, European branch) project to determine the concentration of ergothioneine in 17 their plasma. There was a weak association between the ergothioneine levels and maternal age, but 18 none for BMI. Of these 432 women, 97 went on to develop pre-term (23) or term (74) pre-eclampsia. 19 If a threshold was set at the 90 th percentile of the reference range in the control population (≥ 462 20 ng/mL), only one of these 97 women (1%) developed pre-eclampsia, versus 97/432 (22. 5%) whose 21 ergothioneine level was below this threshold . One possible interpretation of these findings , con- 22 sistent with previous experiments in a reduced uterine perfusion model in rats, is that ergothioneine 23 may indeed prove protective against pre-eclampsia in humans. An intervention study of some kind 24 now seems warranted. 25

pre-eclampsia; prevention; ergothioneine; metabolomics; nutraceutical; mushrooms 26 27 1. Introduction 28 Pre-eclampsia (PE) is a multi-system disorder of pregnancy, characterized by gesta- 29 tional hypertension and the new-onset of proteinuria and/or another maternal organ dys- 30 function [1-3]. It is widely considered to develop in two stages [4] [5]: an initial poor plac- 31 entation [6] followed by oxidative stress and inflammation [7, 8] . As with many other 32 chronic, inflammatory diseases [9], there is also substantial evidence for the involvement 33 of a microbial component [10, 11]. PE can affect 3-5% of pregnancies worldwide [12], and 34 has the potential to be life -threatening; the only real clinical recourse currently available 35 the premature delivery of the fetus. Early markers for predictin g pre-eclampsia are thus 36 highly desirable. 37 Although some soluble protein markers such as sFlt1 and PlGF have proven to be of 38 value (e.g. [13, 14]), a number of studies have developed the idea that small molecules (i.e. 39 metabolomics [15, 16]) have the potential to provide both diagnostic information, and, via 40 mechanistic reasoning, potentially treatments [17-29]. 41 Ergothioneine is an important antioxidant nutraceutical (commonly derived from the 42 dietary intake of mushrooms) [30-34], for which humans have evolved at least one trans- 43 porter [35, 36]. In one large scale metabolomics study [37] it was by some distance the 44 molecule most associated with the prevention of adverse cardiovascular outcomes. Its lev- 45 els have also been linked with lower ed incidences of cognitive defects [38] , and i t 46 Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. prevented some of the main symptoms in the rat RUPP model of pre-eclampsia [39]. In 47 view of the above, and its status as a potent antioxidant, it was thus considered plausible 48 to have some utility in the prevention and/or diagnosis of PE in humans [40]. 49 The international Screening for Pregnancy Endpoints ( SCOPE) study [25, 41- 43] 50 (http://scopestudy.net) bio-banked early pregnancy blood samples from a large cohort of 51 first time preg nant women, 4.9% whom developed pre -eclampsia as defined [44] by the 52 International Society of the Study of Hypertension in Pregnancy [41]. Pre-term pre- ec- 53 lampsia was defined as disease necessitating delivery before 27 weeks’ gestation. The 54 SCOPE study therefore provided the opportunity to assess whether there was any relation 55 between their levels of ergothioneine and the likelihood of developing early or late -onset 56 pre-eclampsia. The present study reports on a secondary analysis of ergothioneine levels 57 in a previously reported case-control study assessing a panel of metabolite biomarker can- 58 didates for pre-eclampsia risk assessment at 15 +/-1 week of gestation [25]. Women with 59 the highest level of ergothioneine had a significantly reduced likelihood of deve loping 60 either preterm or term pre -eclampsia. One interpretation of such data could be that die- 61 tary supplementation with ergothioneine in pregnancy may reduce pre-eclampsia risk, 62 and this is obviously now worth testing. 63 2. Results 64 65 2.1. Overall summary of the cohort studied 66 The nested Case-Control study was defined in the SCOPE – Europe cohort as de- 67 scribed fully in [25], and the demographics (Table 1 of [25]) are not repeated here. For this 68 work, we leveraged calibration data available in the previous study to estimate the er- 69 gothioneine blood levels (ng/mL) in 432 women of whom 335 did not develop PE, 23 suf- 70 fered preterm PE, and 74 suffered from term PE. 71 2.2 Ergothioneine levels in the study population 72 In Figure 1 the distribution of ergothioneine levels in the study-subjects are plotted. 73 Figure 1. Distribution of ergothioneine levels in the study population. 74 75 The levels range from ~ 140 ng/ ml to 998 ng/ml, with a median level of ~ 260 ng/ml. 76 From Figure 1, it is clear that ergothioneine levels are not normally distributed within the 77 study population, but that the long-tailed distribution is skewed to higher concentrations. 78 79 The distribution of ages may be observed in Figure 2, where it may also be seen that 80 there w as a weak yet significant correlation of ergothioneine levels with age (r 2= 0.08; 81 p<0.0001). However, there was no significant relationship (r = - 0.07; p=0.14) between er- 82 gothioneine levels and BMI (Figure 3). 83 84 85 0 5 10 15 20 25 100 220 340 460 580 700 820 940 1060 Ergothioneine (ng/mL) Relative frequency (%) . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 86 87 88 89 Figure 2. Relationship between ergothioneine level and maternal age. 90 91 Figure 3. Lack of relationship between ergothioneine level and BMI. Data have been 92 jiggered in the x-axis direction for clarity 93 94 95 96 Relationship between ergothioneine level and maternal age No PE Preterm PE Term PE [Ergothioneine] ng/mL AGE /y Absence of a relationship between ergothioneine level and BMI BMI /kg.m -2 [Ergothioneine ] No PE Preterm PE Term PE . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 2.3. Levels of ergothioneine in control and in preclamptic women 97 A chief finding here is illustrated in Fig 4 , where we show the relationship between 98 the levels of ergothioneine and whether the women concerned developed pre-eclampsia, 99 whether at term or pre-term. Taking a threshold of >462 ng/mL, which is equivalent to the 100 90th percentile in the control population, we see that only 1/74 developed PE at term, and 101 0/23 developed PE pre-term, i.e. only 1/97 (1%) when these numbers are combined devel- 102 oped any form of PE. 33/335 (10%) who did not develop PE were also above this threshold. 103 In other words, only a single individual out of 432 examples both had a level of ergothi- 104 oneine above 462 ng/mL and developed any form of PE. 105 106 The ergothioneine levels for those three classes are given in Table 1, where it is clear 107 that without considering the specific distributions in detail the mean and median figures 108 would not have indicated an association between ergothioneine and pre -eclampsia risk. 109 From the distributions as plotted in Fig 4 it can be inferred that within the study popula- 110 tion, there is sub-set of study participants who have markedly higher ergothioneine blood 111 levels, giving rise to a bimodal distribution within the study population. 112 The relationship shown in Figure 4 (i.e. setting a cut-off at the 90th centile) was post- 113 hoc, yet it should be evident that a data-driven, machine learning analysis ( to be con- 114 trasted with a frequentist statistical approach [45, 46]) would have discovered it, much as 115 it did [22] in our first foray into pre-eclampsia metabolomics (using a low-resolution mass 116 spectrometer that – as is still common even with high- resolution instruments [47 -50] – 117 could not identify most peaks of interest). We also note that reference- range-based rules 118 are commonly used in clinical diagnostics to identify a population of interest , whereby 119 patients with blood levels of a marker o f interest in e.g. the top or bottom 10% of the ref- 120 erence interval are flagged. 121 Using the above 90th centile as a threshold to identify a population of interest, the 122 following Odds Ratio for developing pre-eclampsia is found in this group ; OR = 0.095 123 with 95% Confidence Interval (0.0129 to 0.706) and significance level p<0.02. 124 125 126 Pregnancy Outcome No Pre-eclampsia Preterm Pre-eclampsia Term Pre-eclampsia N 335 23 74 Ergothioneine (ng/ml) Min – Max 140 – 998 149 – 383 166 -497 Mean (95% CI) 294 (281 – 307) 259 (234 – 294) 276 (259 – 293) Median (95% CI) 260 (248 – 271) 268 (230 – 288) 260 (247 -282) Standard Deviation 123 58 73 25-75th Percentile (IQR) 211 – 332 214 – 290 220 - 333 10-90th Percentile 184 – 462 183 – 340 188 - 382 Normal Distribution (p)* < 0.0001 0.34 0.13 127 128 Table 1. Frequentist statistical analysis of the three pregnancy outcome classes here considered ; * 129 Shapiro- Wilk test; p< 0 .05: Reject Normality; IQR: Inter Quartile Range; 95% CI: 95% Confidence 130 interval 131 132 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 133 Figure 4. Relationship between ergothioneine level and pre-eclampsia category. Data have been jiggered in the y- 134 axis direction for clarity 135 136 137 Figure 5. Violin plots based on individual measurements; --- median levels; blue line- 90th centile based on ergothi- 138 oneine distribution in the control population. Data as in Figure 4 with statistical summary in Table 1. 139 3. Discussion 140 Stimulated by the recognition that ergothioneine might be a potentially useful thera- 141 peutic in PE [39, 40], and that it is certainly a potent (and safe [51]) antioxidant nutraceu- 142 tical [30, 32-34, 52-59], we assessed its concentration in early pregnancy in a representative 143 sample of 1 st time pregnant women who participated in the European branch of the 144 Relationship between ergothioneine level and pre- eclampsia class [Ergothioneine] ng/mL No PE Preterm PE Term PE . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint SCOPE project. Certainly the range of concentrations observed was substantial, from an 145 estimated ~ 140 ng/m l up to 1 µg/ml (Fig 3). Within the study population, the median 146 levels were found to be non-differential between women who developed preterm-, term- 147 pre-eclampsia or women who did not develop pre-eclampsia later in pregnancy. How- 148 ever, within the women who had ergothioneine levels ≥ the 90th percentile of the reference 149 population, i.e., women who did not develop pre -eclampsia, only one study participant 150 developed pre-eclampsia. 151 The levels of ergothioneine reflect both intake (especially from mushrooms [60, 61]) 152 and the activity of the various ergothioneine transporters [35, 36, 62- 64], and neither of 153 these were either known or controlled. Thus, as with a related study on cardiovascular 154 event incidence, where ergothioneine was strongly (indeed the molecule most strongly) 155 associated with more favourable ou tcomes in terms of morbidity and mortality [37], this 156 was a purely observational study. Ergothioneine was also the metabolite associated with 157 the lowest hazard ratio for all-cause mortality [65], and had the third highest loading in a 158 signature collection of healthy metabolites [66] . Mushroom consumption is also strongly 159 associated with a lowering of all- cause mortality [67] and of the incidence of mild cogni- 160 tive impairment [68]. Consequently, since certain aspects of the pre -eclampsia syndrome 161 share hallmarks of vascular disease [69-71], this study adds weight to the idea that it might 162 be a useful nutraceutical in the prevention of (cardio)vascular diseases more generally. 163 4. Materials and Methods 164 Ergothioneine was one of the compounds analysed with multiplex targeted liquid chro- 165 matography – tandem mass spectrometry assay for pre-eclampsia biomarker candidates 166 as detailed in [25]. In the latter study, biomarker levels were expressed as relative concen- 167 trations whereby for any sample the target metabolite read-out was divided by the read- 168 out as obtained from a stable-isotope labelled metabolite internal standard spiked in equal 169 amounts across all samples. Deuterated ergothioneine (D9) served as the stable -isotope 170 labelled metabolite internal standard for ergothioneine quantification. 171 For this secondary data analysis, the relative ergothioneine concentrations were converted 172 in estimated blood levels (ng/ml) using the calibrat ors co-analysed with the patient sam- 173 ples [25]. 174 In brief, calibration curves were generated by mea ns of firstly fortifying a pooled 175 plasma (Technopath plasma with 2% K2-EDTA anticoagulant Lot PF-05171, Technopath, 176 Ireland) with the metabolites of interest and then serially diluting the fortified sample 177 with PBS/BSA buffer (0.01M phosphate buffer and 0.5% Albumin). The metabolites levels 178 for the fortified matrix as well as the concentration span were estimated based on prelim- 179 inary evaluations. This led to the creation of an 8-point calibration curve for all metabolites 180 spanning a ~20-fold dynamic range. ( CAL1; relative level = 100 to CAL8, relative level = 181 5.83). In a separate experiment the levels of ergothioneine in the pooled plasma were de- 182 termined by means of standard addit ion [72], yielding an estimated level of 138 ng/ml. 183 Using this information, the ergothioneine calibration range expressed in ng/mL is easily 184 derived (174 ng/ml (CAL8) – 752 ng/ml (CAL1)). It is noted that said calibration ran ge 185 covers 93.5% of all patient samples assessed; moreover , we typically find that the linear 186 range extends beyond the set calibration range for the metabolites assessed. Verification 187 that the ergothioneine calibrator curves effectively mitigated technical variability fol- 188 lowed the estimation of imprecision from the analysis of 73 duplicate patient samples (in- 189 dependently prepared and randomly distributed across study batches) using the method 190 of Hyslop and White [73], returning a satisfactory Coefficient of Variation (%) = 11.5%. 191

192 The very striking observation that, in this cohort, only 1 individual out of 97 (1%) 193 women with an ergothioneine level abo ve 462 ng/ml manifested pre-eclampsia, whereas 194 in the total cohort 97/432 (22.5%) did, demands an explanation. The easiest one is that this 195 molecule is significantly protective towards (and may be consumed during) the 196 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint development of pre -eclampsia. It is worth noting that, as well as its occurrence in all 197 known culinary mushrooms, ergothioneine is an available nutritional supplement, whose 198 safety has been well estab lished [51, 74-77]. Such an analysis will best be done via a ran- 199 domized controlled trial. 200 201 Author Contributions: Conceptualization, DK, LK and RT; analytical methodology, LB, PO & RT; 202 formal analysis, DK, RT.; investigation, LB, PO, RT, DK; resources; data curation, LK, PO & RT .; 203 writing—original draft preparation, DK, RT & LB ; writing—review and editing, DK, RT & LK .; 204 funding acquisition, LK & RT . All authors have read and agreed to the published version of the 205 manuscript. 206 Funding: This study received funding from the EU -HEALTH Project IMPROvED (305169) of the 207 Seventh Framework Programme, the goal of IMPROvED is to develop a clinically robust predictive 208 blood test for pre-eclampsia. SCOPE was funded by the New Enterprise Research Fund, Foundation 209 for Research Science and Technology; Health Research Council; and Evelyn Bond Fund, Auckland 210 District Health Board Charitable Trust (New Zealand); Premier’s Science and Research Fund, South 211 Australian Government (Australia); and Health Research Board (Ireland). The funders had no role 212 in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 213 DBK is supported by the Novo Nordisk Foundation (grant NNF20CC0035580). 214 Institutional Review Board Statement: As described [25] , Ethics approval was gained from local 215 ethics committees of each participating centre (Manchester, Leeds, and London 06/MRE01/98, Cork 216 ECM5 (10) 05/02/08). Collection of data and biological samples complied with standardised proce- 217 dures in all participating centres and was conducted in accordance with the principles of the Decla- 218 ration of Helsinki. 219 Informed Consent Statement: Written informed consent was obtained from all participants. 220 221 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint Acknowledgments: The investigators thank the pregnant women who participated in the SCOPE 222 study for their indispensable contribution. 223 Conflicts of Interest: DBK is a named inventor on a patent (WO2020221795) describing the produc- 224 tion of ergothioneine in yeast. Leslie W. Brown, Paloma Ortea and Robin Tuytten are employees of 225 Metabolomic Diagnostics, and they and Louise C. Kenny are minority shareholders of Metabolomic 226 Diagnostics. Leslie W. Brown, Robin Tuytten, and Louise C. Kenny are named inventors on a Patent 227 Application which discloses the use of specific proteins and metabolites to predict pre -eclampsia 228 risk, and which is assigned to Metabolomic Diagnostics, i.e., “Methods of predicting pre term birth 229 from pre-eclampsia using metabolic and protein biomarkers”, with publication number 230 WO2019155075A1. 231

232 1. Magee, L. A.; Pels, A.; Helewa, M.; Rey, E.; von Dadelszen, P.; Canadian Hypertensive Disorders of Pregnancy Working 233 Group, Diagnosis, evaluation, and management of the hypertensive disorders of pregnancy. Pregnancy Hypertens 2014, 4, 234 (2), 105-45. 235 2. Rich-Edwards, J. W.; Ness, R. B.; Roberts, J. M., Epidemiology of Pregnancy-Related Hypertension. Chesley's Hypertensive 236 Disorders in Pregnancy, 4th Edition 2015, 37-55. 237 3. Phipps, E. A.; Thadhani, R.; Benzing, T.; Karumanchi, S. A., Pre-eclampsia: pathogenesis, novel diagnostics and therapies. 238 Nat Rev Nephrol 2019, 15, (5), 275-289. 239 4. Redman, C. W. G.; Sargent, I. L., Placental stress and pre-eclampsia: a revised view. Placenta 2009, 30 Suppl A, S38-42. 240 5. Redman, C. W.; Sargent, I. L.; Staff, A. C., IFPA Senior Award Lecture: making sense of pre-eclampsia - two placental 241 causes of preeclampsia? Placenta 2014, 35 Suppl, S20-5. 242 6. Roberts, D. J.; Post, M. D., The placenta in pre-eclampsia and intrauterine growth restriction. J Clin Pathol 2008, 61, (12), 243 1254-60. 244 7. Braekke, K.; Harsem, N. K.; Staff, A. C., Oxidative stress and antioxidant status in fetal circulation in preeclampsia. Pediatr 245 Res 2006, 60, (5), 560-4. 246 8. Manna, S.; Ruano, C. S. M.; Hegenbarth, J. C.; Vaiman, D.; Gupta, S.; McCarthy, F. P.; Mehats, C.; McCarthy, C.; Apicella, 247 C.; Scheel, J., Computational Models on Pathological Redox Signalling Driven by Pregnancy: A Review. Antioxidants 248 (Basel) 2022, 11, (3). 249 9. Kell, D. B.; Pretorius, E., No effects without causes. The Iron Dysregulation and Dormant Microbes hypothesis for chronic, 250 inflammatory diseases. Biol Rev 2018, 93, 1518-1557. 251 10. Kell, D. B.; Kenny, L. C., A dormant microbial component in the development of pre-eclampsia. Front Med Obs Gynecol 252 2016, 3, 60. 253 11. Kenny, L. C.; Kell, D. B., Immunological tolerance, pregnancy and pre-eclampsia: the roles of semen microbes and the 254 father. Front Med Obs Gynecol 2018, 4, 239. 255 12. Mol, B. W. J.; Roberts, C. T.; Thangaratinam, S.; Magee, L. A.; de Groot, C. J. M.; Hofmeyr, G. J., Pre-eclampsia. Lancet 2016, 256 387, (10022), 999-1011. 257 13. Powers, R. W.; Jeyabalan, A.; Clifton, R. G.; Van Dorsten, P.; Hauth, J. C.; Klebanoff, M. A.; Lindheimer, M. D.; Sibai, B.; 258 Landon, M.; Miodovnik, M.; Eunice Kennedy Shriver Natl Inst of Child Health Human Development Matern al-Fetal 259 Medicine Units Network, Soluble fms-Like Tyrosine Kinase 1 (sFlt1), Endoglin and Placental Growth Factor (PlGF) in 260 Preeclampsia among High Risk Pregnancies. Plos One 2010, 5, (10). 261 14. Zeisler, H.; Llurba, E.; Chantraine, F.; Vatish, M.; Staff, A. C.; Sennstrom, M.; Olovsson, M.; Brennecke, S. P.; Stepan, H.; 262 Allegranza, D.; Dilba, P.; Schoedl, M.; Hund, M.; Verlohren, S., Predictive Value of the sFlt-1:PlGF Ratio in Women with 263 Suspected Preeclampsia. N Engl J Med 2016, 374, (1), 13-22. 264 15. Oliver, S. G.; Winson, M. K.; Kell, D. B.; Baganz, F., Systematic functional analysis of the yeast genome. Trends Biotechnol 265 1998, 16, (9), 373-378. 266 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 16. Kell, D. B.; Oliver, S. G., The metabolome 18 years on: a concept comes of age. Metabolomics 2016, 12, (9), 148. 267 17. Austdal, M.; Tangeras, L. H.; Skråstad, R. B.; Salvesen, K.; Austgulen, R.; Iversen, A. C.; Bathen, T. F., First Trimester Urine 268 and Serum Metabolomics for Prediction of Preeclampsia and Gestational Hypertension: A Prospective Screening Study. 269 Int J Mol Sci 2015, 16, (9), 21520-38. 270 18. Bahado-Singh, R. O.; Akolekar, R.; Mandal, R.; Dong, E.; Xia, J.; Kruger, M.; Wishart, D. S.; Nicolaides, K., Metabolomics 271 and first-trimester prediction of early-onset preeclampsia. J Matern Fetal Neonatal Med 2012, 25, (10), 1840-7. 272 19. Chen, T. T.; He, P.; Tan, Y.; Xu, D. Y., Biomarker identification and pathway analysis of preeclampsia based on serum 273 metabolomics. Biochem Bioph Res Co 2017, 485, (1), 119-125. 274 20. Dessì, A.; Marincola, F. C.; Fanos, V., Metabolomics and the great obstetrical syndromes - GDM, PET, and IUGR. Best Pract 275 Res Cl Ob 2015, 29, (2), 156-164. 276 21. Kelly, R. S.; Giorgio, R. T.; Chawes, B. L.; Palacios, N. I.; Gray, K. J.; Mirzakhani, H.; Wu, A.; Blighe, K.; Weiss, S. T.; Lasky- 277 Su, J., Applications of metabolomics in the study and management of preeclampsia: a review of the literature. 278 Metabolomics 2017, 13, (7). 279 22. Kenny, L. C.; Dunn, W. B.; Ellis, D. I.; Myers, J.; Baker, P. N.; The GOPEC Consortium; Kell, D. B., Novel biomarkers for 280 pre-eclampsia detected using metabolomics and machine learning. Metabolomics 2005, 1, (3), 227-234 - online DOI: 281 10.1007/s11306-005-0003-1. 282 23. Kenny, L. C.; Broadhurst, D.; Brown, M.; Dunn, W. B.; Redman, C. W. G.; Kell, D. B.; Baker, P. N., Detection and 283 identification of novel metabolomic biomarkers in preeclampsia. Reprod Sci 2008, 15, (6), 591-7. 284 24. Kenny, L. C.; Broadhurst, D. I.; Dunn, W.; Brown, M.; Francis-McIntyre, S.; North, R. A.; McGowan, L.; Roberts, C.; 285 Cooper, G. J. S.; Kell, D. B.; Philip N Baker on behalf of the SCOPE consortium, Robust early pregnancy prediction of later 286 preeclampsia using metabolomic biomarkers Hypertension 2010, 56, 741-749. 287 25. Kenny, L. C.; Thomas, G.; Poston, L.; Myers, J. E.; Simpson, N. A. B.; McCarthy, F. P.; Brown, L. W.; Bond, A. E.; Tuytten, 288 R.; Baker, P. N.; Screening for Pregnancy Endpoints Consortium, Prediction of preeclampsia risk in first time pregnant 289 women: Metabolite biomarkers for a clinical test. PLoS One 2020, 15, (12), e0244369. 290 26. Koster, M. P. H.; Vreeken, R. J.; Harms, A. C.; Dane, A. D.; Kuc, S.; Schielen, P. C. J. I.; Hankemeier, T.; Berger, R.; Visser, 291 G. H. A.; Pennings, J. L. A., First-Trimester Serum Acylcarnitine Levels to Predict Preeclampsia: A Metabolomics 292 Approach. Dis Markers 2015, 2015, 857108. 293 27. Kuc, S.; Koster, M. P. H.; Pennings, J. L. A.; Hankemeier, T.; Berger, R.; Harms, A. C.; Dane, A. D.; Schielen, P. C. J. I.; 294 Visser, G. H. A.; Vreeken, R. J., Metabolomics profiling for identification of novel potential markers in early prediction of 295 preeclampsia. PloS one 2014, 9, (5), e98540. 296 28. Nobakht, M. G. B. F., Application of metabolomics to preeclampsia diagnosis. Syst Biol Reprod Med 2018, 64, (5), 324-339. 297 29. Woodham, P. C.; O'Connell, T.; Grimes, J.; Haeri, S.; Eichelberger, K.; Baker, A.; Boggess, K., Metabolomics to predict 298 severe preeclampsia in early pregnancy. Am J Obstet Gynecol 2012, 206, (1), S348-S348. 299 30. Borodina, I.; Kenny, L. C.; McCarthy, C. M.; Paramasivan, K.; Pretorius, R.; Roberts, T. J.; van der Hoek, S. A.; Kell, D. B., 300 The biology of ergothioneine, an antioxidant nutraceutical. Nutr Res Rev 2020, 33, 190-217. 301 31. Cheah, I. K.; Halliwell, B., Ergothioneine; antioxidant potential, physiological function and role in disease. Biochim Biophys 302 Acta 2012, 1822, (5), 784-93. 303 32. Cheah, I. K.; Halliwell, B., Ergothioneine, recent developments. Redox Biol 2021, 42, 101868. 304 33. Halliwell, B.; Cheah, I. K.; Tang, R. M. Y., Ergothioneine - a diet-derived antioxidant with therapeutic potential. FEBS Lett 305 2018, 592, 3357-3366. 306 34. Halliwell, B.; Cheah, I., Ergothioneine, where are we now? FEBS Lett 2022, 596, (10), 1227-1230. 307 35. Gründemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.; Lazar, A.; Berkels, R.; Jung, N.; Rubbert, A.; Schömig, E., Discovery of 308 the ergothioneine transporter. Proc Natl Acad Sci 2005, 102, (14), 5256-61. 309 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 36. Gründemann, D.; Hartmann, L.; Flögel, S., The Ergothioneine Transporter (ETT): Substrates and Locations, an Inventory. 310 FEBS Lett 2022, 596, 1252-1269. 311 37. Smith, E.; Ottosson, F.; Hellstrand, S.; Ericson, U.; Orho-Melander, M.; Fernandez, C.; Melander, O., Ergothioneine is 312 associated with reduced mortality and decreased risk of cardiovascular disease. Heart 2020, 106, 691-697. 313 38. Wu, L. Y.; Kan, C. N.; Cheah, I. K.; Chong, J. R.; Xu, X.; Vrooman, H.; Hilal, S.; Venketasubramanian, N.; Chen, C. P.; 314 Halliwell, B.; Lai, M. K. P., Low Plasma Ergothioneine Predicts Cognitive and Functional Decline in an Elderly Cohort 315 Attending Memory Clinics. Antioxidants (Basel) 2022, 11, (9). 316 39. Williamson, R. D.; McCarthy, F. P.; Manna, S.; Groarke, E.; Kell, D. B.; Kenny, L. C.; McCarthy, C. M., L-(+)-Ergothioneine 317 Significantly Improves the Clinical Characteristics of Preeclampsia in the Reduced Uterine Perfusion Pressure Rat Model. 318 Hypertension 2020, 75, 561-568. 319 40. Kerley, R. N.; McCarthy, C.; Kell, D. B.; Kenny, L. C., The potential therapeutic effects of ergothioneine in pre-eclampsia. 320 Free radical biology & medicine 2018, 117, 145-157. 321 41. Kenny, L. C.; Black, M. A.; Poston, L.; Taylor, R.; Myers, J. E.; Baker, P. N.; McCowan, L. M.; Simpson, N. A. B.; Dekker, G. 322 A.; Roberts, C. T.; Rodems, K.; Noland, B.; Raymundo, M.; Walker, J. J.; North, R. A., Early pregnancy prediction of 323 preeclampsia in nulliparous women, combining clinical risk and biomarkers: the Screening for Pregnancy Endpoints 324 (SCOPE) international cohort study. Hypertension 2014, 64, (3), 644-52. 325 42. McCowan, L. M. E.; Thompson, J. M.; Taylor, R. S.; Baker, P. N.; North, R. A.; Poston, L.; Roberts, C. T.; Simpson, N. A.; 326 Walker, J. J.; Myers, J.; Kenny, L. C.; SCOPE consortium, Prediction of Small for Gestational Age Infants in Healthy 327 Nulliparous Women Using Clinical and Ultrasound Risk Factors Combined with Early Pregnancy Biomarkers. PLoS One 328 2017, 12, (1), e0169311. 329 43. Vieira, M. C.; Poston, L.; Fyfe, E.; Gillett, A.; Kenny, L. C.; Roberts, C. T.; Baker, P. N.; Myers, J. E.; Walker, J. J.; McCowan, 330 L. M.; North, R. A.; Pasupathy, D.; Scope Consortium, Clinical and biochemical factors associated with preeclampsia in 331 women with obesity. Obesity (Silver Spring) 2017, 25, (2), 460-467. 332 44. Brown, M. A.; Magee, L. A.; Kenny, L. C.; Karumanchi, S. A.; McCarthy, F. P.; Saito, S.; Hall, D. R.; Warren, C. E.; Adoyi, 333 G.; Ishaku, S.; International Society for the Study of Hypertension in, P., Hypertensive Disorders of Pregnancy: ISSHP 334 Classification, Diagnosis, and Management Recommendations for International Practice. Hypertension 2018, 72, (1), 24-43. 335 45. Breiman, L., Statistical modeling: The two cultures. Stat Sci 2001, 16, (3), 199-215. 336 46. Broadhurst, D.; Kell, D. B., Statistical strategies for avoiding false discoveries in metabolomics and related experiments. 337 Metabolomics 2006, 2, (4), 171-196. 338 47. Dunn, W. B.; Erban, A.; Weber, R. J. M.; Creek, D. J.; Brown, M.; Breitling, R.; Hankemeier, T.; Goodacre, R.; Neumann, S.; 339 Kopka, J.; Viant, M. R., Mass Appeal: metabolite identification in mass spectrometry-focused untargeted metabolomics. . 340 Metabolomics 2013, 9, S44-S66. 341 48. Wright Muelas, M.; Roberts, I.; Mughal, F.; O’Hagan, S.; Day, P. J.; Kell, D. B., An untargeted metabolomics strategy to 342 measure differences in metabolite uptake and excretion by mammalian cell lines. Metabolomics 2020, 16, 107. 343 49. Roberts, I.; Wright Muelas, M.; Taylor, J. M.; Davison, A. S.; Xu, Y.; Grixti, J. M.; Gotts, N.; Sorokin, A.; Goodacre, R.; Kell, 344 D. B., Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and 345 outcome. Metabolomics 2022, 18, 6. 346 50. Shrivastava, A. D.; Swainston, N.; Samanta, S.; Roberts, I.; Wright Muelas, M.; Kell, D. B., MassGenie: a transformer-based 347 deep learning method for identifying small molecules from their mass spectra. Biomolecules 2021, 11, 1793. 348 51. Turck, D.; Bresson, J. L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K. I.; Mangelsdorf, I.; 349 McArdle, H. J.; Naska, A.; Neuhauser-Berthold, M.; Nowicka, G.; Pentieva, K.; Sanz, Y.; Siani, A.; Sjodin, A.; Stern, M.; 350 Tome, D.; Vinceti, M.; Willatts, P.; Engel, K. H.; Marchelli, R.; Poting, A.; Poulsen, M.; Schlatter, J.; Ackerl, R.; van Loveren, 351 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint H.; EFSA Panel Dietetic Prod Nutr, Safety of synthetic L-ergothioneine (Ergoneine (R)) as a novel food pursuant to 352 Regulation (EC) No 258/97. EFSA J 2016, 14, (11). 353 52. Ames, B. N., Prolonging healthy aging: longevity vitamins and proteins. Proc Natl Acad Sci 2018, 115, (43), 10836-10844. 354 53. Beelman, R. B.; Kalaras, M. D.; Phillips, A. T.; Richie, J. P., Jr., Is ergothioneine a 'longevity vitamin' limited in the 355 American diet? J Nutr Sci 2020, 9, e52. 356 54. Beelman, R. B.; Phillips, A. T.; Richie, J. P., Jr.; Ba, D. M.; Duiker, S. W.; Kalaras, M. D., Health Consequences of Improving 357 the Content of Ergothioneine in the Food Supply. FEBS Lett 2021. 358 55. Lam-Sidun, D.; Peters, K. M.; Borradaile, N. M., Mushroom-Derived Medicine? Preclinical Studies Suggest Potential 359 Benefits of Ergothioneine for Cardiometabolic Health. Int J Mol Sci 2021, 22, (6). 360 56. Paul, B. D., Ergothioneine: A Stress Vitamin with Anti-Aging, Vascular and Neuroprotective Roles? Antioxid Redox Signal 361 2021, 36, 16-18. 362 57. Fu, T. T.; Shen, L., Ergothioneine as a Natural Antioxidant Against Oxidative Stress-Related Diseases. Front Pharmacol 363 2022, 13, 850813. 364 58. Whitmore, C. A.; Haynes, J. R.; Behof, W. J.; Rosenberg, A. J.; Tantawy, M. N.; Hachey, B. C.; Wadzinski, B. E.; Spiller, B. 365 W.; Peterson, T. E.; Paffenroth, K. C.; Harrison, F. E.; Beelman, R. B.; Wijesinghe, P.; Matsubara, J. A.; Pham, W., 366 Longitudinal Consumption of Ergothioneine Reduces Oxidative Stress and Amyloid Plaques and Restores Glucose 367 Metabolism in the 5XFAD Mouse Model of Alzheimer's Disease. Pharmaceuticals (Basel) 2022, 15, (6), 742. 368 59. Nakamichi, N.; Tsuzuku, S.; Shibagaki, F., Ergothioneine and central nervous system diseases. Neurochem Res 2022, 47, (9), 369 2513-2521. 370 60. Dubost, N. J.; Beelman, R. B.; Peterson, D.; Royse, D. J., Identification and Quantification of Ergothioneine in Cultivated 371 Mushrooms by Liquid Chromatography-Mass Spectroscopy. Int J Med Mush 2005, 8, 215-222. 372 61. Kalaras, M. D.; Richie, J. P.; Calcagnotto, A.; Beelman, R. B., Mushrooms: A rich source of the antioxidants ergothioneine 373 and glutathione. Food Chem 2017, 233, 429-433. 374 62. Gründemann, D., The ergothioneine transporter controls and indicates ergothioneine activity--a review. Prev Med 2012, 54 375 Suppl, S71-S74. 376 63. Cheah, I. K.; Ong, R. L.; Gruber, J.; Yew, T. S.; Ng, L. F.; Chen, C. B.; Halliwell, B., Knockout of a putative ergothioneine 377 transporter in Caenorhabditis elegans decreases lifespan and increases susceptibility to oxidative damage. Free Radic Res 378 2013, 47, (12), 1036-45. 379 64. Yee, S. W.; Buitrago, D.; Stecula, A.; Ngo, H. X.; Chien, H. C.; Zou, L.; Koleske, M. L.; Giacomini, K. M., Deorphaning a 380 solute carrier 22 family member, SLC22A15, through functional genomic studies. Faseb J 2020, 34, 15734-15752. 381 65. Yan, Y.; Smith, E.; Melander, O.; Ottosson, F., The association between plasma metabolites and future risk of all-cause 382 mortality. J Intern Med 2022. 383 66. Smith, E.; Ericson, U.; Hellstrand, S.; Orho-Melander, M.; Nilsson, P. M.; Fernandez, C.; Melander, O.; Ottosson, F., A 384 healthy dietary metabolic signature is associated with a lower risk for type 2 diabetes and coronary artery disease. BMC 385 Med 2022, 20, (1), 122. 386 67. Ba, D. M.; Gao, X.; Muscat, J.; Al-Shaar, L.; Chinchilli, V.; Zhang, X.; Ssentongo, P.; Beelman, R. B.; Richie, J. P., Jr., 387 Association of mushroom consumption with all-cause and cause-specific mortality among American adults: prospective 388 cohort study findings from NHANES III. Nutr J 2021, 20, (1), 38. 389 68. Feng, L.; Cheah, I. K.; Ng, M. M.; Li, J.; Chan, S. M.; Lim, S. L.; Mahendran, R.; Kua, E. H.; Halliwell, B., The association 390 between mushroom consumption and Mild Cognitive Impairment: a community-based cross-sectional study in 391 Singapore. J Alzheimers Dis 2019, 68, 197-203. 392 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint 69. Conti, E.; Zezza, L.; Ralli, E.; Caserta, D.; Musumeci, M. B.; Moscarini, M.; Autore, C.; Volpe, M., Growth factors in 393 preeclampsia: a vascular disease model. A failed vasodilation and angiogenic challenge from pregnancy onwards? 394 Cytokine Growth Factor Rev 2013, 24, (5), 411-25. 395 70. Leon, L. J.; McCarthy, F. P.; Direk, K.; Gonzalez-Izquierdo, A.; Prieto-Merino, D.; Casas, J. P.; Chappell, L., Preeclampsia 396 and Cardiovascular Disease in a Large UK Pregnancy Cohort of Linked Electronic Health Records: A CALIBER Study. 397 Circulation 2019, 140, (13), 1050-1060. 398 71. Vogel, B.; Acevedo, M.; Appelman, Y.; Bairey Merz, C. N.; Chieffo, A.; Figtree, G. A.; Guerrero, M.; Kunadian, V.; Lam, C. 399 S. P.; Maas, A.; Mihailidou, A. S.; Olszanecka, A.; Poole, J. E.; Saldarriaga, C.; Saw, J.; Zuhlke, L.; Mehran, R., The Lancet 400 women and cardiovascular disease Commission: reducing the global burden by 2030. Lancet 2021, 397, (10292), 2385-2438. 401 72. Kruve, A.; Rebane, R.; Kipper, K.; Oldekop, M. L.; Evard, H.; Herodes, K.; Ravio, P.; Leito, I., Tutorial review on validation 402 of liquid chromatography-mass spectrometry methods: part II. Anal Chim Acta 2015, 870, 8-28. 403 73. Hyslop, N. P.; White, W. H., Estimating precision using duplicate measurements. J Air Waste Manag Assoc 2009, 59, (9), 404 1032-9. 405 74. Schauss, A. G.; Vértesi, A.; Endres, J. R.; Hirka, G.; Clewell, A.; Qureshi, I.; Pasics, I., Evaluation of the safety of the dietary 406 antioxidant ergothioneine using the bacterial reverse mutation assay. Toxicology 2010, 278, (1), 39-45. 407 75. Forster, R.; Spézia, F.; Papineau, D.; Sabadie, C.; Erdelmeier, I.; Moutet, M.; Yadan, J. C., Reproductive safety evaluation of 408 L-Ergothioneine. Food Chem Toxicol 2015, 80, 85-91. 409 76. Marone, P. A.; Trampota, J.; Weisman, S., A safety evaluation of a nature-identical L-ergothioneine in Sprague Dawley 410 rats. Int J Toxicol 2016, 35, (5), 568-83. 411 77. Turck, D.; Bresson, J. L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K. I.; Mangelsdorf, I.; 412 McArdle, H. J.; Naska, A.; Neuhauser-Berthold, M.; Nowicka, G.; Pentieva, K.; Sanz, Y.; Siani, A.; Sjodin, A.; Stern, M.; 413 Tome, D.; Vinceti, M.; Willatts, P.; Engel, K. H.; Marchelli, R.; Poting, A.; Poulsen, M.; Schlatter, J. R.; Ackerl, R.; van 414 Loveren, H.; EFSA Panel Dietetic Prod Nutr, Statement on the safety of synthetic L-ergothioneine as a novel food - 415 supplementary dietary exposure and safety assessment for infants and young children, pregnant and breastfeeding 416 women. EFSA J 2017, 15, (11). 417 418 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.19.22283617doi: medRxiv preprint