Itaconic acid is an organic compound with the formula . With two carboxyl groups, it is classified as a dicarboxylic acid. It is a non-toxic white solid that is soluble in water and several organic solvents. It plays several roles in biology.
Reactions Upon heating, itaconic acid converts to its anhydride.
As a dicarboxylic acid, itaconic acid has two pKa values. At pH levels above 7, itaconic acid exists as its double negatively charged form, termed itaconate.
As an α,β-unsaturated carbonyl compound, itaconic acid is a good electrophile for Michael reactions. Thus, nucleophiles add across the C=C bond. (R = organic group). This reaction is the means by which the fire retarding chemical 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide can be incorporated into polymers.
Production In 1836, Samuel Baup discovered itaconic acid as a by-product in a dry distillation of citric acid. In the late 1920s, itaconic acid was isolated from a fungus in the Aspergillus genus of fungi The dry distillation forms itaconic anhydride, which then is hydrolyzed. Since the 1960s, however, it has been produced commercially by fermenting glucose, molasses, or another abundant carbon sources by a fungus such as Aspergillus itaconicus, Aspergillus terreus, or Ustilago maydis have also been investigated. One generally accepted route by which fungi make itaconate is through the tricarboxylic acid cycle pathway. This pathway forms cis-aconitate which is converted to itaconate by cis-aconitate-decarboxylase. Animal cells also make itaconate by an enzyme-catalyzed reaction from cis-aconitate, an intermediate metabolite in the tricarboxylic acid cycle, (i.e., TCA cycle). The itaconate-producing reaction is stimulated when the TCA cycle is suppressed.
Ustilago maydis makes itaconic acid from trans-aconitate, catalyzed by aconitate delta-isomerase. The trans-aconitate product is decarboxylated to itaconate by trans-aconitate decarboxylase (i.e., TAD1, an enzyme found in Ustilago maydis) Itaconate has also been obtained by fermenting the fungi Yarrowia lipolytica with glucose, various species of Candida fungi with glucose, Ustilago vetiveriae fungus with glycerol, and various species of Aspergillus niger fungi with glucose, sorbitol, or sorbitol plus xylose mixture. Fermenting Escherichia coli bacteria with glucose, xylose, glycerol, or starch and Corynebacterium glutamicum bacteria with glucose or urea also affords itaconic acid. Ustilago maydis has, however, been genetically engineered to increase its itaconic acid production,
History In the 1930s itaconate was shown to have bactericidal actions. In 2011, Strelko et al. reported that itaconate was produced by two mammalian immortalized cell lines, cultured mouse VM-M3 brain tumor cells and RAW 264.7 mouse macrophages, and by macrophages isolated from mice. This group also showed that stimulation of mouse macrophages with the bacterial toxin, lipopolysaccharide (i.e., LPS, also termed endotoxin), increased their production and secretion of itaconate. In 2013, Michelucci et al. revealed the biosynthesis pathway that makes itaconate in mammals. These publications were followed by numerous others focused on the biology of itaconate and certain itaconate-like compounds as regulars of various cellular responses in animals and possibly humans.
Biology of Itaconate Biological studies focus on itaconate's physiological and pathological functions.
Cellular sources of itaconate The major cell types that normally make itaconate in response to stressful conditions or inflammatory stimuli are phagocytes of the myeloid lineage. This includes macrophages, monocytes, dendritic cells, and neutrophils. Itaconate is also produced by certain myeloid-derived suppressor cells (MDSCs). In the absence of inflammatory stimuli (i.e., under homeostatic conditions) itaconate production is low. Phagocyte stimulation leads to upregulation of IRG1 (immune-responsive gene 1), which encodes the enzyme required to synthesize itaconate.
Itaconate-forming metabolic pathway Itaconate is a by-product of the tricarboxylic acid cycle, consisting of eight successive enzyme-catalyzed biochemical reactions that occur in the cell's mitochondria. When cis-aconitate, accumulates, aconitate decarboxylase (also termed ACOD1, cis-aconitate decarboxylase) metabolizes cis-aconitate to itaconate and carbon dioxide (CO2) in the following decarboxylation reaction:
cis-aconitate → itaconate + CO2
This itaconate is transported across the mitochondrial membrane into the cell's cytosol by the mitochondrial dicarboxylate carrier protein, mitochondrial 2-oxoglutarate/malate carrier protein, and citrate–malate shuttle. The cytosolic itaconate may then move form the cytosol through the patients' cells' surface membranes to the extracellular space (this trans-membrane movement may involve a specific transport protein such as the major facilitator superfamily transport protein (i.e., MfsA) in fungi.) This itaconate has mostly anti-inflammatory actions. It acts on its parent cell, other cells, and certain microorganism by stimulating or inhibiting the activity of various response-regulating pathways in its parent cell, other cells, and bacteria. Itaconate's actions on its parent and other cells were considered as entirely independent of any receptor. Itaconate stimulates certain mammalian cells by activating the OXGR1 receptor.
OXGR1 receptor OXGR1 (also known as GPR99) is a G protein-coupled receptor that was identified in 2004 as a receptor for the tricarboxylic cycle intermediate, α-ketoglutarate. In 2013, it was found to also be a receptor for leukotriene E4 and to lesser extents leukotriene C4 and D4. Among a set of cultured human embryonic kidney HEK 293 cells made to express any one of 351 different human G protein-coupled receptors, only cells expressing OXGR1 responded to itaconate by raising their cytosolic Ca2+ levels. HEK 293 cells expressing any of the other 350 receptors did not consistently alter their cytosolic Ca2+ levels in response to itaconate. Respiratory epithelium cells isolated from control mice (i.e., these cells naturally express OXGR1) but not from Oxgr1 gene knockout mice (i.e., these cells lacked OXRG1) responded to itaconic acid by raising their cytosolic Ca2+ levels and stimulating their mucociliary clearance (equivalent to stimulating the secretion of mucus). Application of itaconate in the noses of control mice but not Oxgr1 gene knockout mice stimulated nasal secretion of mucus. Oxgr1 gene knockout mice and Irg1 gene knockout mice (mice lacking the itaconate-producing protein, IRG1) that were intranasally infected with Pseudomonas aeruginosa had increased numbers of these bacteria in their lung tissue and bronchoalveolar lavage fluid (i.e., airway washing) than control mice that respectively expressed OXGR1 and IRG1. α-ketoglutarate and itaconate, which have similar structures, activate OXGR1-expressing HEK293 cells at similar concentrations, i.e., between 200 and 300 μM/liter. These findings indicate that itaconate stimulates human HEK 293 and mouse respiratory epithelial cells by activating their OXGR1 receptors. Since OXGP1 is expressed in a wide range of tissues and mediates the allergic and inflammatory responses to the cited leukotrienes, it may be involved in the inflammatory responses detailed in the following "Actions of itaconate and its analogs" section. That is, itaconate, like succinate (see previous paragraph), may stimulate cells by receptor-dependent and receptor independent mechanisms. Future studies need to determine the extent to which OXGR1 contributes to the various actions of itaconate and itaconate-like compounds (see next section) as well as the potencies of each of these agents in activating OXGR1.
Itaconate and itaconate-like compounds 4-Octyl itaconate, dimethyl itaconate, and 4-ethyl itaconate have been used to mimic the biological effects of itaconate. These functional analogs of itaconate are often used in place of itaconate because of their presumed greater ability to pass through the surface membranes of, and thereby enter, cells. Many studies have examined the actions of itaconate analogs rather than itaconate itself and that itaconate and these analogs have on occasion shown significantly different biological activities.
The anionic forms of mesaconic and citraconic acids, i.e., mesaconate and citraconate, are isomers of itaconate that differ from itaconate by the location of their internal carbon to carbon double bonds (i.e., C=C). The two isomers have some but not all of the biological activities of itaconate. (Meconate is a natural product made by mouse macrophages.) Other compounds have been synthesized that enter cells and then breakdown into itaconate plus a second inflammation-inhibiting agent, carbon monoxide. These compounds, termed itaCORMs, activate some of the anti-inflammatory pathways activated by itaconate but also to have the anti-inflammatory activity of carbon monoxide in suppressing production of the pro-inflammatory cytokine, interleukin-23. The itaCORMs require further study. Analyses of itaconate as well as each of the itaconate analogs, itaconate isomers, and itaCORM may be useful for selecting the agent(s) best suited to treat the human disorders which preclinical studies suggest are improved by itaconate or an itaconate-like compound(s).
Dietary sources of itaconate and its isomers Itaconic acid and its two isomers, mesaconic and citraconic acids, were found in rye and wheat breads with appreciably higher concentrations of itaconic and citraconic acids in their crusts (i.e., outer bread layer) than crumbs (i.e., soft inner part of the bread). Based on the average consumption of bread and bread-related baked goods in Germany, the daily intake of itaconate plus its two isomers was estimated to be from 7 to 20 micrograms. Rats have been shown to absorb the itaconic acid that was added to their diet.
Actions of itaconate and its analogs Itaconate and its analogs can operate concurrently through multiple pathways to induce their effects. Relevant to this, future studies must determine the role of the newly defined receptor for itaconate, OXGR1, in contributing to the mediation of the following actions of itaconate and itaconate-like compounds.
Inhibit succinate dehydrogenase Succinate dehydrogenase (i.e., SDH) is an enzyme complex of six proteins in the mitochondrial tricarboxylic acid cycle that metabolizes succinate to fumarate. (Although bacteria generally lack mitochondria, their surface membranes have a similar SDH system.) Itaconate inhibits SDH's activity thereby blocking succinate's oxidation to fumarate and causing succinate levels to increase. Itaconate has been reported to increase succinate levels in a wide variety of cells including cultured mouse RAW264.7 macrophages, macrophages differentiated from human monocytes, Huh7 human liver carcinoma cells, human MCF-7 breast cancer cells, human A549 lung adenocarcinoma cells, and the brain neurons and astrocytes generated from rat embryo brain tissue. This succinate stimulates various responses in its parent and other cells as detailed elsewhere (see SUCNR1 and succinic acid).
Inactivate KEAP1