Ergothioneine as an Emerging Food-Derived Bioactive Compound Protecting Against Age-Related Diseases: Issues Needing More Research

1. Introduction

Ergothioneine (ET; structure shown in Figure 1) is a compound first discovered in 1909; it was thought until recently that it can only be made by fungi and some bacteria, including cyanobacteria, and a few yeasts [1,2,3,4,5,6,7,8,9,10,11]. However, a recent report [12] suggests that ET can also be made by certain mosses and gymnosperm plants, although not by angiosperms, a category that includes most plants eaten by humans, such as asparagus, corn, wheat and rye [12]. An interesting evolutionary question would be to understand why angiosperms lost the capacity to make ET during their evolution [12], a topic mentioned again in Section 6 below.

Several researchers investigated the properties of ET in the 1940s and 1950s, mostly studying its antioxidant abilities. It was then largely ignored for decades, as reflected by the low number of published papers recorded on PubMed (Figure 2). Melville [8] provided an excellent review of this early work. When humans consume ET, it is rapidly absorbed by a selective transporter (OCTN1, organic cation transporter novel 1, often now called the ET transporter, ETT) present in the small intestine (and in many other organs; Figure 3), distributed to all body tissues, avidly retained (half-life in the body estimated at several weeks) and renally reabsorbed; very little is excreted in urine but some in faeces (discussed further in Section 6 below) [1,2,3,4,9,13,14]. ET also passes from mother to child [2,3,9,15,16] since it is present in breast milk and the mammy gland shows increased levels of OCTN1 during lactation [9]. In addition, OCTN1 has been detected in human placenta (reviewed in ref [17], also see ref [18]). These facts strongly suggest that ET is important to the human body: selective transporters are usually only present to take up essential vitamins (e.g. vitamin C [19]) and minerals such as iron [20]; many other dietary compounds with antioxidant properties (e.g. most polyphenols) are poorly absorbed and rapidly metabolised, so that their concentrations in human tissues and body fluids are very low (reviewed in [2,21,22]). By contrast, blood and tissue concentrations of ET in humans and other animals are usually much higher [1,2,3,4,21,22,23,24,25]; some specific values for ET concentrations in humans are given in Section 3 below. Indeed, this potential importance of ET was recognised as long ago as 1959, by Melville [8], who wrote “the avidity with which dietary ET is incorporated into tissues of all animals, the tenacity with which it is held there, and its characteristic non-uniform pattern of distribution in these tissues (see Figure 3) are all facts which hint that there is a purpose in its presence”.

2. Where Does ET Come from in the Human Diet?

The major dietary source of ET in most populations is mushrooms [6,7,9,10,11,22,23,24,25], although smaller amounts are found in other foods such as asparagus, corn, wheat, other plants, Spirulina, meat, fish, eggs, milk and even Beluga whales [6,7,9,10,11,22,23,24,25,26,27,28,29]. Feeding hens with the ET-producing fungus Neurospora crassa was observed to increase the ET content of their eggs [29]. To quote references [23] and [30], “Ergothioneine intake, which derives almost totally from mushrooms, was evaluated in different countries and age groups, such as Belgium (average intake of 0.062 mg/kg body weight/day in children), France (0.03 mg/kgbw/day in adults), Ireland (0.053 mg/kgbw/day in adults), Italy, Finland (0.069 mg/kgbw/day in adults), and the United States. Higher average intakes were reported for Italian children and adults, with both being 0.067 mg/kg bw/day. On the other hand, the lowest intakes were reported for US adults and children, with average daily consumption of ergothioneine at 0.016 mg/kg bw/day and 0.014 mg/kg bw/day, respectively” [23,30]. Children were defined as age range 2.5–6.5 years, adults defined as over 18 (Italy and Ireland), 15 (France) or 25 (Finland) [30]. Beelman et al [31] attributed the high Italian intakes to frequent consumption of ET-rich Porcini mushrooms. Dietary intakes across the various regions of China varied widely, with a mean value estimated as 0.043 mg/kgbw/day [32]. By contrast, in the Inuit population, who appear to have exceptionally high blood ET concentrations (Section 3 below), the main sources of ET seem to be meat and marine foods including Beluga whales [26,27,28].

3. Why Has Interest in ET Increased?

The recent growing interest in ET, as revealed by more publications (Figure 2), was sparked by two factors. One was the identification of OCTN1, encoded by the gene slc22a4, as the ET transporter [33,34,35]. ET is highly water soluble [as well as tasteless and odourless] so a transporter is needed to take it across the hydrophobic interior of cell membranes. OCTN1 can transport a few other substrates in vitro [17,36] but studies of its transport kinetics reveal that ET is likely to be the major, if not the only, substrate in vivo [33,34,35,37,38]. Hence, OCTN1 is often now simply referred to as the ET transporter [34,35]. However, OCTN1 can also transport selenoneine [39], an analogue of ET in which the sulphur is replaced by selenium [Se]. Selenoneine is found mostly in foods of marine origin, although there have been few studies as yet on its role in vivo and its concentrations in most populations are much lower than those of ET [26,27,28,39].

A second factor raising interest in ET was the growing realisation, first pointed out in 2016 [40,41], that low blood/plasma/serum ET concentrations in humans are strongly associated with an increased risk of developing age-related diseases such as cognitive impairment, cardiovascular disease, dementia, frailty, Parkinson’s disease and eye disorders, including cataract and macular degeneration [2,3,4,22,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58] (Figure 4), as well as erectile dysfunction [59], chronic renal disease [60] and pre-eclampsia [61]. Indeed, ET has been suggested to be a compound critical to implantation of the fertilised egg into the uterus wall and hence for pregnancy maintenance [62]. As an illustration, Figure 4 shows the range of plasma ET concentrations in an Asian (Singaporean, mostly Chinese) population [2,42,43,44]; a concentration below 810 nanomolar (nM), 0.81 micromolar (µM), is associated with increased risk of age-related diseases. Similarly, in an Australian population median serum ET levels were 0.79-1.35 µM but 24% lower in subjects with psychological stress [54]. Most ET in human blood is present in erythrocytes, which can have millimolar (mM) intracellular ET concentrations (reviewed in [63]), so whole blood concentrations of ET are much greater than plasma levels. For example, in a healthy Singaporean population mean plasma ET concentration was about 1µM whereas whole blood levels were around 152 µM [14]. Similarly, in an Arab (Saudi Arabian) population whole blood levels were around 122-161µM [64]. Although different research groups have measured plasma, serum or whole blood ET concentrations in humans, we [40,56] and others [65] have shown that plasma, serum, whole blood and erythrocyte levels are strongly correlated. By contrast, whole blood ET concentrations in an Inuit population were reported as around 400 µM whereas the control group (laboratory members in Quebec, Canada) had whole blood concentrations close to 100µM [28], broadly similar to the Saudi, Australian and Singaporean populations described above [14,64].

Of course, correlations between disease incidence and ET concentrations, however strong, do not prove causation, as explained in Figure 5. However, a cause-consequence relationship between low ET concentrations and the above age-related diseases is strongly suggested by, first, the predictive value of low ET concentrations in identifying increased disease risk or disease progression (e.g. refs [47,58,59]. For example, in a Singaporean population, healthy subjects with low plasma ET concentrations [Figure 4] are at increased risk of developing cognitive impairment, dementia and other diseases [2,40,42,43,44]. Similarly, in a cohort of Japanese subjects aged 69-79, the mean serum ET levels were 0.639µM (below the “healthy threshold”, Figure 4) and 273 of the 1344 subjects went on to develop dementia [45].

Second, further evidence for a cause-consequence relationship is provided by a large and growing number of studies on laboratory animals, human induced pluripotent stem cells (iPSC), primary neurons, other cell types, organoids, brain slices and other systems showing that ET is highly neuroprotective in models of dementia, stroke, eye disease, hearing loss, neuropathy, depression, sleep disorders and Parkinson’s Disease, among others (reviewed in [1,2,3,22,44], also see [66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90]). Several pilot human clinical trials have indicated significant neuroprotective effects of ET [91,92,93]. For example, in the pilot study by Yau et al [91] administration of ET (25 mg 3 times per week) for a year or more to Asian subjects aged 60 or above with mild cognitive impairment (MCI) raised mean plasma ET concentrations from around 0.8 µM to about 3.96 µM, improved cognitive function, and prevented increases in the plasma concentrations of neurofilament light protein, a biomarker of neuronal damage. In the study by Zajac et al [93] in an Australian population, consumption of ET (10mg or 25mg daily) raised plasma levels from a median of 1.154 µM to 9 µM (10mg ET daily) or 16 µM (25mg ET daily) after 16 weeks and improved sleep and some aspects of cognition. Many other clinical trials with ET are underway, including a trial on kidney dialysis patients, who have low ET levels because washout of ET during dialysis is aggravated by its impaired reabsorption by the dysfunctional kidneys [94,95]. For example, in a United States (USA) cohort [96] subjects on full dialysis had mean ±SD plasma ET concentrations of 0.47 ± 0.20 µM compared with controls of 2.1 ± 1.6 µM. Peritoneal dialysis subjects had concentrations of 0.67 ± 0.30 µM ET [96]. Both levels would seem to fall in the “at risk” range (Figure 4). Indeed, it has been suggested that patients with chronic kidney disease should eat more mushrooms [97].

As a result of the above discoveries, ET is becoming increasingly popularised on social media, podcasts, and talk shows as a “healthy longevity” agent. The number of published papers on it, although still small (e.g. as compared to papers on the possible health benefits of flavonoids, carotenoids or vitamins C and E), is rapidly increasing (Figure 2), although overall awareness of ET among the public and medical professionals is still low. ET is now being sold by itself, or as a constituent of several “longevity supplements”, through multiple online platforms. Pure ET appears safe for human consumption and has been approved for human usage by the European and USA Food and Supplement Safety Regulatory Agencies (reviewed in [2,3]). Of course, a major problem revealed by analyses of multiple commercial supplements of different nutrients is poor quality control (QC); a recent example reports that many of the formulations of the popular supplement nicotinamide mononucleotide (NMN) being sold do not match the contents claimed on the labels [98]. Hence, ET for human use should be obtained from a reputable source that has established and validated (ideally externally validated) QC protocols.

Although the number of published papers describing ET is increasing (Figure 2), many of them are reviews or meta-analyses (for some recent examples see [99,100,101,102,103,104,105,106]) and many new ones continue to appear. Very few of these publications, and indeed few research groups globally, are investigating in depth the fundamental biochemistry and molecular biology underlying the actions of ET, and their relationship to human health and nutrition. This lack of basic research into ET is unfortunate and needs to be addressed. Let us discuss the areas that the author believes need attention.

4. Ergothioneine Chemistry and Metabolism Have Scarcely Been Explored, Especially in the Brain

The chemistry of ET (Figure 1) in relation to biological systems has only been explored to a very limited extent [107,108,109], although all agree that ET is a water-soluble, tasteless, odourless and chemically-stable molecule that resists heat and does not degrade on prolonged storage, allowing its accurate analysis in stored biological materials. However, repeated freeze-thawing of biological samples should be avoided as limited loss of ET may sometimes occur (our unpublished data and ref [110]. This stability helps to facilitate the use of ET as a food preservative [111].

Several studies report that ET’s unique chemical properties can improve the light-harvesting efficiency of solar cells [112,113,114]—but the mechanism(s) by which this occurs and their possible relevance to biological systems have not been elucidated. Three metabolites of ET have been identified [2,3], namely hercynine, ET sulphonate and S-methyl ET (Figure 1) but there has been no systematic search for others. S-methyl ET presumably arises by the action of a methyltransferase, but this enzyme has not been identified. None of these three compounds is present in mice lacking OCTN1, who have no ET in their tissues [2], suggesting that all three derive from ET. A role for ET in drug metabolism has been suggested: incubation of liver microsomes from several animals with ET and NADPH led to production of ET conjugates with added paracetamol, raloxifene, diclofenac, clozapine, carbamazepine, and tamoxifen, among other drugs [115,116]. The significance of this in vivo remains to be discovered, although a raloxifene—ET conjugate has been detected in vivo in rats [115].

It has been suggested that when ET acts as an antioxidant by scavenging various oxygen radicals and other reactive oxygen species (ROS, a term explained in detail in ref [21]) the sulphonate and hercynine are produced, and thus could be biomarkers of ROS generation [117,118,119], i.e. their concentrations in plasma or serum might provide evidence that ET has antioxidant properties in vivo (discussed further in section 5 below). However, this needs to be validated in vivo in humans by comparison with established biomarkers of ROS generation and oxidative damage, as reviewed in [14,21,120,121]. Oxidation products of ET exposed to various ROS in vitro also include a thiyl radical (ES^—), ET disulphide and 5-oxoET (Figure 1) [108,109]. The enzymes that act upon ET in humans and other animals and how ET influences the cell and tissue lipidomes, metabolomes and transcriptomes have not been elucidated, although some preliminary proteomic and metabolomic studies have been published in rodents [122,123]. Some of the oxidized forms of ET can be reduced by the enzyme glutathione reductase plus its co-substrate reduced glutathione (GSH) or by thioredoxin reductase enzymes [109] but the importance of this in vivo needs more investigation. ET has also been reported to inhibit γ-glutamyl transpeptidase, an enzyme on cell surfaces that catalyses the hydrolysis of GSH, although fairly high concentrations of ET are required (K_i ~170 µM) [124]. A claim that nM concentrations of ET increase the activities of certain human carbonic anhydrase enzymes [125] remains to be confirmed. Nor have the potential biological roles of the three known ET metabolites (Figure 1) (and any other metabolites yet to be identified) been investigated. Do they contribute to ET’s cytoprotective effects? When ET enters cells, some of it is taken up by mitochondria [5,126], but where the rest of it goes is unknown, nor has the subcellular distribution of OCTN1 been accurately mapped, apart from its presence on plasma membranes [35]. Nor has ET’s mechanism of entry into mitochondria been elucidated [126]. OCTN1 appears to be involved, but there seem to be additional transporter(s).

This lack of knowledge of ET distribution, metabolism and any biological effects of its metabolites is especially true for the brain. ET has been reported to be present in human and other animal brains (and in human cerebrospinal fluid) in multiple studies [2,3,9,14,44,48,65,127,128,129,130,131,132,133,134], and orally supplementing laboratory animals with ET increases their brain levels of this compound [130,132]. However, no one has yet identified OCTN1 in intact human blood-brain barrier [2,35] although it is present in human brain microvascular endothelial cells and several other regions of the human brain (Figure 3) (reviewed in [2,35]. It thus remains to be discovered how ET enters the human brain, as it clearly does.

OCTN1 is not the only molecule capable of transporting ET. Another human transporter, SLC22A15, encoded by the slc22a15 gene, which is not involved in the intestinal absorption of ET but is widespread in the brain (Figure 3), can also transport ET, albeit with much lower affinity (K_m ~ 400 µM) and lower V_max compared with OCTN1, K_m ~ 21 µM [9,35,135,136]; the major substrate of SLC22A15 seems to be creatine [137]. Mitochondria from OCTN1-knockout mice can still take up ET, albeit at a slower rate than mitochondria from wild-type mice, and the additional transport mechanism(s) involved have not yet been identified [126].

5. The Mechanism(s) of Action of Ergothioneine Is(Are) Unclear and Are Likely to Be Multifactorial

ET is frequently described as an antioxidant, although determinations of its redox potential (0.45 volts) show it to be a weaker reducing agent than GSH, cysteine or ascorbate [138]. Nevertheless, in vitro studies have shown that ET is capable of scavenging several biologically-important ROS, including hydroxyl radical (^—OH), hypochlorous acid (HOCl), hypobromous acid (HOBr), ozone (O₃), peroxynitrite (ONOO⁻/ONOOH), singlet oxygen [118,139,140,141,142,143] and the pro-oxidant higher haem oxidation states (ferryl haem) of haemoglobin and myoglobin [144,145]. ET can also render pro-oxidant transition metal ions (Fe²⁺, Cu⁺) less redox active by chelating them [139,146]. It can scavenge methylglyoxal, acrolein, and possibly other cytotoxic aldehydes that result from lipid peroxidation [147,148].

In addition to direct scavenging of ROS, several studies have presented evidence that ET can activate the “master regulator” of endogenous antioxidant defences, Nrf2, and so raise endogenous antioxidant defence levels in vivo [71,149,150,151,152,153,154,155]. However, how ET activates Nrf2 is unclear: does it increase the phosphorylation of keap1 through activation of various kinase enzymes [150,153,155,156,157,158], or does ET interact directly with the thiol groups on keap1, or both; these are the two usual mechanisms by which Nrf2 is activated [157,159]? The role of these two established mechanisms of Nrf2 activation needs to be elucidated for ET, although in silico studies have suggested that ET might bind to the active site of Nrf2 to prevent its proteasomal degradation and thereby allow it to promote the transcription of genes encoding antioxidant defences [152].

Despite its demonstrated antioxidant abilities, evidence that ET exerts significant antioxidant effects in vivo in humans remains limited, as discussed in [2,14], although if ET only acts as an antioxidant at specific sites where it accumulates in the human body, this would be hard to detect by measuring systemic biomarkers of oxidative stress (reviewed in [21,120,121,159]. Indeed, tissue injury, which usually leads to increased oxidative damage for a variety of reasons, as reviewed in [21,160], can lead to increases in the levels of OCTN1, resulting in higher concentrations of ET in the injured tissue [161,162]. This elevation of OCTN1 has been proposed to be an adaptive cytoprotective mechanism, bringing in more ET to protect the injured tissue [161,162], although any protection by the extra ET need not necessarily be by antioxidant action, of course.

Several other lines of evidence suggest that the antioxidant properties of ET may not be its only, or even its major, mechanism of action in vivo. First, antioxidant supplements have generally shown only limited effectiveness in the treatment or prevention of human diseases [21,159,163,164,165,166,167,168] whereas ET seems more promising (as reviewed in Section 3 above), although this remains to be fully validated by large-scale double blind placebo-controlled clinical trials. Second, ET biosynthesis has been identified in anaerobes, suggesting that ET production may have evolved for reasons other than antioxidant properties, although no metabolic roles of ET specific to anaerobes have yet been discovered [169,170,171]. However, this is not a conclusive argument, because it remains possible that anaerobes evolved antioxidants (including ET perhaps) to protect against free radicals generated by radiation damage, which was a major problem in the “pre-oxygen” world because of the lack of Earth’s ozone layer [21,170]). Indeed, several papers have described radioprotective effects of ET [172,173,174]. These antioxidants might have later allowed anaerobes to survive transient exposures to O₂ as O₂ levels rose in the atmosphere due to the evolution of photosynthesis [21,175,176,177]. In any case, these studies [169,170,171] indicate that ET had an early evolutionary origin, perhaps as an antioxidant, but as life evolved it may have taken on other roles. Interestingly, ET has been shown to play a role in biosynthesis of lincosamide antibiotics (such as lincomycin A) in a few microorganisms [178,179].

Indeed, multiple mechanisms of ET action other than antioxidant ones have been suggested (Figure 6) and their relative importance has not been elucidated. It may, of course, vary depending on the organism, organ, tissue, cell, organelle and the injurious agent. For example, the protective effect of ET against the development of fatty liver in a mouse model was attributed to inhibition of expression of the enzyme phosphatidylethanolamine cytidyltransferase 2, thus decreasing the accumulation of toxic phosphatidylethanolamine species [180]. The anti-inflammatory and anti-fibrotic properties of ET described in multiple animal studies and attributed to variety of mechanisms [60,61,76,88,156,158,181,182,183,184,185,186,187,188,189,190,191] may be of especial importance in prevention and treatment of age-related diseases, including neurodegenerative diseases, in which inflammation is intimately involved, as it is in ageing generally (“inflammaging”) [192,193]. The proposed abilities of ET to promote neurogenesis and neuronal differentiation and enhance the actions of neurotrophic factors [194,195,196,197,198,199] may also be very important in protection against and treatment of neurodegeneration. A role in early childhood brain development is also possible, since ET passes from mother to baby [2,3,9,15,16]. To date, these studies are mostly based on work with cell cultures, although human studies are beginning to appear [198]—more studies are needed to establish the importance of ET in vivo in enhancing neurogenesis, synaptogenesis, neuronal differentiation and the beneficial effects of neurotrophic factors, and the mechanisms by which ET is acting. It cannot be over-emphasised that, as proposed in [44], a brain-penetrant agent safe for human consumption, that not only slows neurodegeneration but also promotes the genesis of new neurons and supports the functions of new and existing neurons, would be of immense value in the slowing and prevention of age-related diseases of the nervous system, especially for dementia, in the world’s rapidly-ageing populations; time will tell if ET can deliver on this potential. This is especially true given the growing evidence that neurogenesis and neuronal development contribute to cognitive resilience in the brains of patients with Alzheimer’s disease [199,200] and ET may help to promote cognitive resilience [58,201].

Established mechanisms (good evidence) Antioxidant (ROS scavenging, radioprotection, scavenging of cytotoxic aldehydes, chelation of iron and copper ions to prevent them catalyzing oxidative damage, Nrf2 activation) Anti-inflammatory (including microglia) Protection of mitochondrial structure and function (morphology, ATP synthesis, mitochondrial DNA)

Likely Mechanisms (evidence is accumulating) Promotes neurogenesis Enhances action / promotes synthesis of neurotrophic factors Stimulates neuronal differentiation Restores long term potentiation (a key component of memory) Clearing amyloid / protecting against amyloid toxicity Generates H2S Affects macrophage polarization / cytokine production (e.g. [156,285] Raises NAD+ levels, stimulates sirtuin activity Interactions with the microbiome

Suggested mechanisms (evidence for a role in vivo is limited as yet) Inhibits ferroptosisa Prevents cell senescenceb Alters the epigenome by modulating DNA methylation/demethylation [157] Affects autophagyc

Comments:

• Ferroptosis is a mode of cell death that involves iron ions and lipid peroxidation [286], both of which play key roles in oxidative damage in vivo [21,160,286,287]. Preliminary studies in vitro suggest that ET can inhibit ferroptosis (Chester Drum, Irwin Cheah and Ong Wei Yi, personal communications).
• Several papers have suggested that ET might inhibit cell senescence [61,288,289], a term which refers to persistent cell cycle arrest due to stressful events such as telomere shortening, oxidative damage, activation of oncogenes etc [290]. However, ET failed to exert anti-senescence effects in a model of replication – stress associated senescence (Prof. Marie-Veronique Clement and Dr. Le Luo, personal communication, also ref [291]), or in etoposide-induced cellular senescence (Prof. Rachel Watson and Drs Keith Tan, Oliver Dreesen and Selwyn Loh, personal communication). In the cases where ET did protect, it seemed to be acting by diminishing oxidative stress [288,289].

Several papers have suggested that ET could modulate autophagy [61,189,292] but the importance of this in vivo and the mechanism(s) by which ET could do this are unclear as yet.

Returning to the mechanisms of action of ET, two papers published in Cell Metabolism in 2025 proposed that the actions of ET in mitochondria are its major cytoprotective role, suggesting that the generation of hydrogen sulphide (H₂S) from ET is a key mechanism [122,202]. However, they reached different conclusions as to how this is achieved. One paper [122] reported that ET binds to and activates the enzyme 3-mercaptopyruvate sulphotransferase, but the other [202] that ET acts as a substrate for cystathionine gamma-lyase, leading to increases in NAD⁺ concentrations. Indeed, there is a voluminous literature claiming that raising tissue NAD⁺ concentrations promotes healthy longevity [98,203] although there are some conflicting data (reviewed in [203,204]). These discrepancies between references [122] and [202] need to be resolved. In any case, is H₂S likely to explain all the biological actions of ET? This could be the case, but this author is sceptical. Low levels of H₂S can indeed be neuroprotective [205,206,207,208,209,210,211], but higher H₂S levels can cause tissue damage [210,212,213,214,215], as illustrated by studies on breast cancer [215], human stroke and animal models of stroke [212,213]. Indeed, to quote a recent paper [214] on the mechanisms used by the nematode C. elegans to avoid exposure to H₂S: “H₂S is an energy source, a toxin and a gasotransmitter”. The biological effects of the widely used “antioxidant” drug N-acetylcysteine (NAc) have also been suggested, at least in part, to be due to H₂S generation [121,216,217], yet NAc has both protective and damaging effects in vivo, including promoting cancer development under some circumstances (reviewed in [159]). By contrast, no one has yet found pro-neurodegenerative or other toxic effects of ET, even at high doses, in any animal model or in the clinical trials conducted to date [2,3].

6. Interaction of ET with the Human Microbiome Has Scarcely Been Studied

It is not only humans and other animals that avidly take up ET; several (and possibly all, most have not been studied) higher plants do so as well, absorbing ET generated by fungi and bacteria in the soil through their roots [6,218,219,220]. Was the loss of ET biosynthesis in higher plants described in the Introduction (Section 1) perhaps related to the ease with which angiosperms can obtain it from fungi and bacteria in the soil [6,218,219,220], saving them the energy cost of making it? This is perhaps analogous to the argument that early humans lost the capacity to make vitamin C because they ate a diet rich in natural fruits and vegetables [21,22]. Hence, it has been suggested that modern agricultural practices that disrupt fungi and bacteria in soil could decrease amounts of ET in dietary plants, leading to potential health risks if body ET concentrations fall too low [6,219,220]. The ET content of plants grown hydroponically is also likely to be low or zero.

Many bacteria also have transporters that allow them to take up and accumulate ET [221,222,223,224,225,226]. ET is efficiently absorbed in the small intestine, but some passes through to the colon and small amounts can be measured in human faeces [2] although very little in urine even after supplementation, probably due to efficient renal reabsorption [14,227]. Presumably if subjects consume ET-rich diets or supplements containing ET, more will end up in the colon. Since the body will have absorbed the ET it requires in the small intestine, uptake and metabolism of ET by the colonic microbiome would not be expected to deprive the human body of ET, but there are other potential concerns. Research into interactions of ET with the microbiome is still in its early stages [58,228,229,230,231], but one concern that has been raised is that some colonic bacteria can degrade ET using the enzyme ergothionase to eventually produce trimethylamine (TMA), which is absorbed from the gut and converted by the liver to trimethylamine oxide (TMAO) [232,233,234]. TMAO is, epidemiologically, the exact opposite of ET: in most (but not all) studies high plasma concentrations of TMAO are associated with increased risk of developing several diseases, including cognitive impairment and cardiovascular disease [232,233,234]. Hence could too much dietary ET be deleterious by raising TMAO levels? Evidence from the absence of increases in plasma TMAO levels in human subjects supplemented with ET, and the lack of correlations of circulating ET and TMAO concentrations in large patient cohorts, suggest that ET is not a significant source of TMAO in humans [93,235], which is consistent with the literature on the safety and health-promoting aspects of ET reviewed above. Another study even suggested that TMA, the precursor of TMAO, is beneficial to human health [236]. Although increased expression of genes encoding ergothionase has been reported in the colonic microbiome of patients with colorectal cancer [230], ET actually appears to be toxic to colorectal cancer cells, based on in vitro studies [237]. It would be good to confirm this action in vivo. Nevertheless, other pathways of ET metabolism by the colonic microbiome are rapidly being discovered [58,229,230,231,238,239] and the potential health effects of the products of these pathways, whether beneficial or deleterious, is an area ripe for further investigation.

The stomach and small intestine also have a microbiome [240,241]. The duodenal/small intestinal microbiome is poorly characterised and how it might take up or metabolise ET and thus affect its uptake into the human body is largely unknown. However, one constituent of the small intestine microbiome is Lactobacillus reuteri (Limosilactobacillus reuteri); strains of this organism have been widely promoted as probiotics to promote human gut health [242,243]. Some strains of L. reuteri avidly take up ET [221] – whether or not these bacteria could absorb enough ET to compete with its human intestinal update is unknown. It presumably would depend on their abundance in vivo; something to think about when selecting probiotics, perhaps – should we even preload them with ET to prevent them taking it from our diet and possibly (given the cytoprotective effects of ET in bacteria described in Section 7 below) to promote their survival in the gastrointestinal tract?

However, an important component of the gastric microbiome is Helicobacter pylori [240,244]. H. pylori cannot synthesize ET, but takes it up avidly, and ET helps to protect this pathogen against the antimicrobial effects of ROS generated by human phagocytes as part of the immune response [222,223,224,225,244]. Indeed, the high efficiency of ET uptake was illustrated in a recent paper [245] describing conjugation of an anti-microbial compound with ET as a vehicle to deliver that compound to H. pylori and eradicate this pathogen. Along similar lines, an ET-coated imaging agent has been used to image kidney disease: the agent bound to OCTN1 in the kidney [246]. Unfortunately, H. pylori can trigger chronic gastritis, in the worst-case scenario leading to gastric cancer [244]. Could uptake of dietary ET by H. pylori aggravate this? We do not know – it has not been studied. As discussed in Section 5 above, ET has anti-inflammatory effects, which should help to ameliorate risk. Epidemiological evidence suggests that consumption of mushrooms, a major dietary source of ET, is negatively associated with risk of gastritis and gastric cancer [247,248]. Of course, mushrooms contain multiple compounds that could be protective against cancer [247,248] and so these studies do not prove that ET is responsible for the protection. An interesting recent observation is that consumption of antacids by a Caucasian population in Rotterdam was associated with worse cognition, correlated with lower plasma ET levels [58]. Raising gastric pH increases the size of the gastric microbiome [240] – perhaps the various organisms can then absorb sufficient ET in the stomach to reduce its bioavailability?

7. ET and Other Human Pathogens

Several bacteria can themselves synthesise ET, and some are human pathogens, including Burkholderia pseudomallei, the causative agent of human melioidosis, a serious and sometimes lethal infection by this organism [249]. One of the most studied ET-synthesising pathogens is Mycobacterium tuberculosis, the agent responsible for human tuberculosis (TB). This organism synthesises two sulphur-containing compounds, ET and mycothiol, to help protect itself against ROS generated by the human immune system, thereby hindering its eradication and facilitating its persistence in the human body [250,251,252]. Indeed, ET concentrations may have increased during evolution of M. tuberculosis to enhance its persistence [250]. Indeed, there has been some work on inhibitors of ET synthesis as potential therapeutic agents [253,254]. Could consuming ET facilitate TB development in infected subjects? M. tuberculosis and other mycobacteria do not possess an uptake transporter for ET, so presumably they cannot accumulate it from their surroundings [223]. However, B. pseudomallei can (ref [249] and our unpublished results). Several mycobacteria, including M. tuberculosis, release some of the ET that they synthesise into their surrounding environment, suggesting that external ET could help to protect them against ROS generated by phagocytes as part of the human immune response [255,256,257]. In addition, H. pylori, Streptococcus pneumoniae, Listeria monocytogenes, Clostridium difficile, Enterococcus faecalis and Staphyloccus aureus are among the pathogenic bacteria that can take up ET and use it for cytoprotection [223,225]. Could dietary ET enhance the pathogenicity of these organisms and of M. tuberculosis by raising ET levels in body fluids so as to protect them against ROS generated by the immune system? Another gap in our knowledge.

8. Can ET Be Synthesized in the Human Body?

The consensus view, based on multiple old and a few recent experiments, is that ET cannot be made in animals [1,2,3,4,8,13,258,259], and our experiments with laboratory mice (reviewed in [2]) confirm that view. All known ET biosynthetic pathways begin with the amino acid histidine [260], so we simply fed mice with isotopically-labelled histidine (N¹⁵-histidine) and looked for N¹⁵-labelled ET, by mass spectrometry, which we never convincingly found. However, bioinformatic analyses of the gut microbiota in humans and other animals confirm that all the genes necessary to make ET are present [221] but of course they may not be being transcribed and translated to produce active enzymes, or present together in a single organism. A recent paper [260,261] suggested that some gut microorganisms (Cyanobacteria and related species) in mice could make ET, although biosynthesis was not directly demonstrated and such bacteria are quite rare in the human gut microbiome [262,263] – more research on this is needed.

However, the human gut not only has a microbiome, it has a mycobiome, a collection of fungi [264,265]. Although fungi are usually less than 0.1% of detected microbial reads or genes in the human gut, they can exert effects on the body by producing bioactive metabolites and interacting with bacteria and the host immune system [265]. Many fungi [including mushrooms of course!] can make ET [8,9,13,266]; could this ever happen in vivo and, if so, would the fungi retain ET or release it into the human body? We don’t know, but a few studies suggest that ET might sometimes be made. For example, ET can be synthesized by several established pathogenic fungi that can reside in the human body, including Cryptococci and Candida species and Aspergillus fumigatus [266,267,268,269]. Could they perhaps sometimes generate and release ET wherever in the human body the fungus is present? The author believes that it is more likely that they will keep it to themselves, but it could be released when the fungi die, e.g. as a result of treatment with anti-fungal medications.

9. Why Some Humans Have Low ET Levels Is Unclear

Low plasma/serum/whole blood ET concentrations in humans are strongly associated with higher risk of age-related diseases, as reviewed in Section 3 above (also see Figure 4). But why do some people have low levels? An obvious explanation is a diet low in sources of ET (hence the frequent suggestion to eat more mushrooms), but evidence suggests that there is more to it than that, e.g. in a Singaporean population with low ET no change in mushroom consumption was observed [40]. For example, poor kidney function [e.g. due to diabetes] could impair reabsorption of ET from the renal ultrafiltrate [94,95,96,187]. The slc22a4 gene that encodes OCTN1 has several polymorphisms [17,35,270,271,272,273,274,275,276,277], that could lead to amino acid substitutions which affect the transport of ET by OCTN1 [271,272]. In our study of ET uptake by healthy young Singaporean subjects [14], we found that plasma ET concentrations after supplementation were higher in subjects who already had higher plasma ET concentrations, suggesting variations in intestinal uptake efficiency. The population frequency in different countries and precise effects of these polymorphisms on the kinetics of ET transport in vivo remain to be studied in detail, one of the many important aspects of ET biology that need to be explored in more depth. For example, the 503F variant of OCTN1 has a lower K_m for ET but also a lower maximal transport velocity (V_max), resulting in a 50% higher initial transport capacity of ET at low concentrations (≤ 10µM) [271] but perhaps less effective transport at higher ET concentrations. Some other variants were reported to have impaired ET transport capacity [272]. Such kinetic studies need to be extended to all the variants, and their frequencies in different populations examined. In addition, a mutation in slc22a4 has been linked to human hearing loss [273,276]. This mutation was reported to severely decrease the efficiency of ET uptake by OCTN1 [273]. Indeed ET has been shown to protect against age-related hearing loss [68] and hearing loss due to treatment with the chemotherapeutic agent cis-platin [151], both in mice, suggesting a role of ET in auditory function.

Many other aspects of ET biology have been hinted at in the literature. For example, a mouse study reported that there are circadian changes in the intestinal expression of OCTN1 that could influence ET uptake from diet [278] and a rat study suggested that administration of 1α,25-dihydroxyvitamin D₃ can modulate OCTN1 levels [279]; the relevance of either to humans has not been studied. The interaction of ET with other dietary nutrients, and their potential effects on ET uptake in the gastrointestinal tract or by the body tissues, have also not been studied. Nor have possible interactions of ET with medications been examined in detail: an early suggestion that ET could influence the pharmacokinetics of the drug gabapentin does not seem to be a significant effect [280,281]. One possible drug interaction could be with the calcium channel blocker verapamil, which inhibits OCTN1 [33] and is often used in the laboratory to examine the role of this transporter in the actions of ET (e.g. [37,70,76]. An effect of antacids on ET concentrations has been described [59], as discussed in Section 6 above. Another issue worth further exploration is species differences. For example, OCTN1 expression is high in rat and mouse livers and human foetal liver [35], but much lower in adult human liver (Figure 3). When mice consume ET, a lot enters the liver [130], but this may not be true in humans [35,282]. The distributions of OCTN1 in the various cell types of mouse and human brain are also different [35].

10. Conclusion

Evidence is rapidly growing that ET is a valuable component of the human diet and may even be essential for healthy longevity [1,2,3,4,8,42,44]; the late Professor Bruce Ames called it a “longevity vitamin” [283]. Indeed, increased OCTN1 activity has been proposed as an early evolutionary trait in Neolithic farmers to decrease the risk of ET deficiency, since many of the plants they first domesticated were low in ET [284]. There is a threshold blood level of ET, below which the risk of multiple age-related diseases increases [2] (Figure 4), but is the optimal level of ET needed to maintain health greater than this? An analogy could perhaps be with vitamin C; only low intakes are needed to prevent overt symptoms of deficiency (scurvy), but larger amounts of vitamin C seem necessary for optimal health [19]. So should we all be consuming ET supplements, or just eating more mushrooms? Many aspects of ET biology need to be investigated before making firm recommendations. I hope that this narrative review will stimulate more research in the field. In studies of a very large number of human subjects, we have never found anyone completely lacking ET: does this mean that OCTN1 and ET are essential for early human development and human life?