Understanding biomarkers

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. Tryptophan → 5-Hydroxytryptophan TPH1, TPH2 · BH4, Iron Tryptophan hydroxylase attaches an OH group to tryptophan. The enzyme carries iron and works with tetrahydrobiopterin (BH4) and oxygen. Two forms exist: TPH1 in the gut, TPH2 in nerve cells. This step is the slowest of the route. It sets the pace. Source 1, 7↑ supplies 5-HTP is the precursor just one step away from serotonin. Because tryptophan hydroxylase works slowly and needs BH4, iron and oxygen, this step determines how much serotonin can form at all. established physiology Source 1, 7
   ⚖ When the balance tips

   too much — If more 5-HTP forms, AADC quickly converts it on to serotonin; it hardly accumulates, because the following step runs faster than this one.

   too little — If little BH4 is available, tryptophan hydroxylase works more slowly and less 5-HTP forms; the same BH4 is also needed by the hydroxylase for dopamine and for making nitric oxide.

   established physiology · Source 1, 2, 7

2. 5-Hydroxytryptophan → Serotonin AADC · Vitamin B6 (PLP) The enzyme AADC removes a carboxyl group from 5-HTP, using pyridoxal phosphate, the active form of vitamin B6. Serotonin is formed separately in two places: in the enterochromaffin cells of the gut lining and in nerve cells of the brain. Source 2, 3↕ both, depending on amount Serotonin is a messenger in many places: in the gut it stimulates movement of the gut wall, in vessels it narrows or widens them, in the brain it carries signals between nerve cells. Depending on the receptor, the same amount excites or dampens. established physiology Source 3, 11
   ⚖ When the balance tips

   too much — If serotonin builds up in the gap between cells, more receptors are occupied for longer; monoamine oxidase then breaks down more of it, and more 5-HIAA appears in the urine.

   too little — If little serotonin is available, the 5-HT receptors are occupied less often, and the pineal gland has less starting material for melatonin.

   established physiology · Source 11, 6

3. Serotonin → N-Acetylserotonin AANAT · Acetyl-CoA In the pineal gland, the enzyme AANAT attaches an acetyl group to serotonin at night. The amount of this enzyme follows the day-night rhythm. Light slows AANAT, which makes melatonin a night-time signal. Source 4↑ supplies N-acetylserotonin is the direct precursor of melatonin. Because AANAT is made in large amounts at night and broken down quickly in light, this step sets when melatonin forms. established physiology Source 4
   ⚖ When the balance tips

   too much — If AANAT runs fully at night in darkness, a lot of N-acetylserotonin forms; ASMT converts it to melatonin, and the night-time rise is high.

   too little — If light falls on the retina at night, AANAT is slowed; little N-acetylserotonin then forms, and the night-time rise in melatonin is smaller.

   established physiology · Source 4

4. N-Acetylserotonin → Melatonin ASMT · SAM The enzyme ASMT transfers a methyl group from SAM to N-acetylserotonin. Melatonin leaves the pineal gland and spreads through the body via the blood. Its rise in the blood is the night-time signal. Source 4↑ supplies Melatonin is the time signal of the night: its level in the blood rises with darkness and falls in the morning. Via MT1 and MT2 it tells the inner clock and many tissues that it is night, and lowers body temperature slightly. established physiology Source 4
   ⚖ When the balance tips

   too much — If there is a lot of melatonin in the blood during the day, the night signal arrives at the wrong time; the inner clock then shifts, forward or backward depending on the timing.

   too little — If the night-time rise is small, for example with light at night, the inner clock receives a weaker night signal, and the day-night rhythm of the tissues is tuned less clearly.

   established physiology · Source 4

5. Melatonin → Melatonin receptors Melatonin binds the receptors MT1 and MT2. They sit, among other places, in the suprachiasmatic nucleus, the inner clock of the brain, and on vessels and further tissues. Through them, the night signal reaches the inner clock. Source 4↑ supplies Via MT1 and MT2, melatonin acts as a night signal: at the inner clock MT1 dampens the nerve cells and MT2 shifts the clock's timing; on vessels they narrow or widen them depending on the type. established physiology Source 4
   ⚖ When the balance tips

   too much — If the receptors are strongly occupied at the wrong time of day, MT2 shifts the inner clock; depending on the timing, it then runs earlier or later.

   too little — If the receptors are occupied little at night, the signal to the inner clock stays weak, and the nerve cells there are dampened less.

   established physiology · Source 4

6. 6-Hydroxymelatonin → 6-Sulfatoxymelatonin Sulfotransferase A sulfotransferase attaches a sulphate group to 6-hydroxymelatonin. In this water-soluble form, the compound leaves the body in the urine. Its amount reflects night-time melatonin formation. Source 4↓ depletes The sulphate group makes the breakdown product water-soluble so that the kidney can excrete it. The amount in the urine over one night reflects how much melatonin was formed during that time. established physiology Source 4
   ⚖ When the balance tips

   too much — If a lot of melatonin was formed at night, a lot of 6-sulphatoxymelatonin also appears in the urine in the morning; the compound itself no longer acts as a time signal.

   too little — If little melatonin was formed at night, for example with light at night, less 6-sulphatoxymelatonin is found in the urine in the morning.

   established physiology · Source 4

7. 5-HT receptors → Reuptake SERT The transporter SERT carries serotonin from the gap between cells back inside. The signal ends there; the same protein also brings serotonin into blood platelets. The faster SERT works, the shorter serotonin acts. Source 11↓ depletes SERT ends the serotonin signal by bringing the messenger back from the gap; inside the cell it is reused or broken down by MAO. The faster SERT works, the shorter serotonin acts at the receptors. established physiology Source 11
   ⚖ When the balance tips

   too much — If SERT works faster, serotonin is cleared from the gap more quickly, and the receptors are occupied for a shorter time.

   too little — If SERT is inhibited, serotonin stays in the gap longer and occupies the receptors longer; the platelets then absorb less serotonin.

   established physiology · Source 11, 3

Further stations

• Tryptophan — amino acid from food Tryptophan is an amino acid the body does not form itself. From dietary protein it enters the blood and follows three routes there: the kynurenine pathway, the route to serotonin and melatonin, and the bacterial route to indole. The routes share one pool. Source 3, 5, 8↑ supplies Tryptophan is the starting material for serotonin and melatonin. It shares its pool with the kynurenine pathway, protein synthesis and gut bacteria; if one of these routes is used more, less remains for the others. established physiology Source 5, 10, 3
  ⚖ When the balance tips

  too much — More tryptophan in the blood does not bring unlimited serotonin: at the blood-brain barrier it shares the transporter with other large amino acids, and the hydroxylase is the slowest step. Much of it runs into the kynurenine pathway.

  too little — If little tryptophan arrives, several routes share a smaller pool; in the brain the hydroxylase then has less starting material, and less serotonin forms.

  established physiology · Source 10, 5, 1

• Kynurenine pathway — second route of tryptophan Most tryptophan does not head towards serotonin but into the kynurenine pathway. There, the enzymes TDO and IDO open the ring part of the molecule. The more this pathway runs, the less tryptophan remains for serotonin. Source 5↓ depletes The kynurenine pathway draws off most of the tryptophan. The harder TDO in the liver or IDO in immune cells work, the less tryptophan remains for serotonin and melatonin. established physiology Source 5, 9
  ⚖ When the balance tips

  too much — If cells make more IDO in response to inflammatory signals, more tryptophan runs into this pathway; tryptophan in the blood falls, and less starting material remains for serotonin formation.

  too little — If TDO and IDO run more slowly, more tryptophan stays in the blood and more of it is available for protein synthesis and serotonin; the formation of NAD⁺ from tryptophan then declines.

  established physiology · Source 9, 5

• 5-HIAA — 5-hydroxyindoleacetic acid Monoamine oxidase (MAO) breaks serotonin down via an aldehyde intermediate. An aldehyde dehydrogenase turns this into 5-HIAA, which is excreted in the urine. This ends serotonin's action. Source 6↓ depletes The formation of 5-HIAA ends serotonin's action: MAO breaks it down, and the breakdown product no longer acts at the receptors. The amount in the urine reflects how much serotonin the body has broken down in total. established physiology Source 6, 3
  ⚖ When the balance tips

  too much — If a lot of serotonin is formed, for example in the gut, more also runs through MAO and more 5-HIAA appears in the urine. The reaction also produces hydrogen peroxide and an aldehyde, which the cell has to break down further.

  too little — If MAO works more slowly, serotonin persists longer and occupies its receptors for longer; less 5-HIAA forms.

  established physiology · Source 6, 3

• 6-Hydroxymelatonin — in the liver In the liver, the enzyme CYP1A2 attaches an OH group to melatonin. In this way the liver clears melatonin quickly from the blood. Source 4↓ depletes The OH group is the first step by which the liver clears melatonin from the blood. Because CYP1A2 works quickly, melatonin stays in the blood only briefly, and its level closely follows formation in the pineal gland. established physiology Source 4
  ⚖ When the balance tips

  too much — If CYP1A2 works faster, melatonin is cleared from the blood more quickly, and the night signal is shorter.

  too little — If CYP1A2 works more slowly, for example because other substances occupy the enzyme, melatonin stays in the blood longer, and the night signal lasts longer.

  established physiology · Source 4

• Indole route — by gut bacteria Bacteria in the large intestine split tryptophan with the enzyme tryptophanase. Indole and related substances arise; they bind the aryl hydrocarbon receptor of the gut lining and leave the body in urine. This branch draws on the tryptophan pool. Source 8↕ both, depending on amount The indole route diverts tryptophan in the gut before it reaches the blood. Indoles act via the aryl hydrocarbon receptor on the gut lining and its immune cells; in animal models they thus keep the mucosal barrier tight. observed in studies Source 8
  ⚖ When the balance tips

  too much — If bacteria convert a lot of tryptophan to indole, less of it is available for uptake into the blood. In the liver, indole is converted to indoxyl sulphate, which is excreted via the kidney.

  too little — If the gut bacteria make little indole, the aryl hydrocarbon receptor of the gut lining receives less signal; in animal models the mucosal barrier then becomes more permeable.

  observed in studies · Source 8

• Serotonin in blood — stored in blood platelets Most of the serotonin comes from the gut. Blood platelets absorb it through the transporter SERT and store it. Serotonin does not cross the blood-brain barrier; the brain forms its own. At injuries, the platelets release it. Source 3↑ supplies Blood platelets collect serotonin and release it when they attach to an injured vessel wall. There it narrows small vessels and attracts further platelets; in this way it contributes to stopping bleeding. established physiology Source 3, 11
  ⚖ When the balance tips

  too much — If the gut releases a lot of serotonin into the blood, the platelets absorb more of it until their store is full; what remains free in the blood is broken down via MAO to 5-HIAA.

  too little — If the platelets contain little serotonin, for example when SERT is inhibited, they release less of it at an injury; the narrowing of small vessels and the attraction of further platelets are weaker.

  established physiology · Source 3, 11, 6

• 5-HT receptors — binding sites Serotonin acts through a family of receptors, 5-HT1 to 5-HT7. Most pass the signal on through G proteins; 5-HT3 is an ion channel. They sit in the gut wall, on vessels and in nerve cells. The receptor decides whether it excites or dampens. Source 11↕ both, depending on amount The receptor decides what effect serotonin has: some types dampen the cell, others excite it, and 5-HT3, as an ion channel, lets ions flow in directly. The same amount of serotonin can thus excite in one place and slow things in another. established physiology Source 11
  ⚖ When the balance tips

  too much — If the receptors are strongly occupied, the processes they control run more strongly: the gut wall moves faster, vessels narrow or widen, and nausea arises via 5-HT3.

  too little — If the receptors are occupied little, these signals are weaker; in the gut, for example, the wall moves more slowly.

  established physiology · Source 11, 3

Cofactors in this pathway

• Tetrahydrobiopterin (BH4) — Cofactor of tryptophan hydroxylase, which forms 5-HTP; with little BH4, less 5-HTP forms Source 1, 7
• Iron — Metal in the active site of tryptophan hydroxylase; without iron this step stops Source 1In the ORY catalogue as a laboratory value: Eisen
• Oxygen — Tryptophan hydroxylase builds an oxygen atom into the ring of tryptophan; without it no 5-HTP Source 1
• Vitamin B6 — As pyridoxal phosphate, cofactor of AADC, which converts 5-HTP to serotonin; with little PLP, less serotonin forms Source 2In the ORY catalogue as a laboratory value: Vitamin B6
• Acetyl-CoA — Supplies AANAT with the acetyl group for N-acetylserotonin; the step that light slows Source 4
• S-adenosylmethionine (SAM) — Methyl group donor for ASMT, which forms melatonin; the last step to melatonin Source 4
• Vitamin B2 (FAD) — As FAD, cofactor of monoamine oxidase, which breaks down serotonin; this ends the serotonin signal Source 6

What acts on this pathway

• Inflammatory signals — Interferon-γ, TNF and bacterial LPS make many cells form more of the enzyme IDO. More tryptophan then enters the kynurenine pathway, and less remains for this step. Source 9, 5
• Competition at transport — Tryptophan shares the transporter LAT1 at the blood-brain barrier with other large neutral amino acids. How much reaches the brain depends on the ratio of these amino acids in the blood. Source 10
• Light at the eye — Light on the retina reports through the suprachiasmatic nucleus, the inner clock, and a nerve pathway to the pineal gland. Less of the enzyme AANAT is then formed, and the nightly rise of melatonin turns out smaller. Source 4

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. Tryptophan → Kynurenine IDO, TDO · Haem (iron), Oxygen The enzymes IDO and TDO open the ring of tryptophan; kynurenine forms via a short-lived intermediate. The kynurenine-to-tryptophan ratio describes how active this step currently is. In immune cells, kynurenine binds the aryl hydrocarbon receptor. Source 1, 2, 8↕ both, depending on amount IDO belongs to immune defence: the cell consumes tryptophan and withholds it from pathogens and from T cells that depend on it. Through the aryl hydrocarbon receptor, kynurenine damps the immune cell further. The same brake spares tissue and weakens defence. established physiology Source 7, 2
   ⚖ When the balance tips

   too much — When IDO runs harder, the kynurenine-to-tryptophan ratio rises. More kynurenine then binds the aryl hydrocarbon receptor in immune cells, and the cell damps its own response.

   too little — When IDO stays quiet, mainly hepatic TDO breaks tryptophan down to kynurenine; more is left for protein building and serotonin. Pathogens and T cells that depend on it then find more of it.

   established physiology · Source 7, 2

   Field of research — The kynurenine-to-tryptophan ratio is studied in inflammation and in disorders of the nervous system. Source 9, 8

2. Kynurenine → 3-Hydroxykynurenine KMO · FAD, NADPH Kynurenine 3-monooxygenase (KMO) attaches an OH group to kynurenine. The enzyme sits in the outer membrane of the mitochondria and works with FAD and NADPH. In cell culture it forms radicals with iron and copper. Source 3, 1↕ both, depending on amount 3-Hydroxykynurenine is a transit compound and an oxidant in one: it can release electrons and form oxygen radicals in doing so. In cell culture, accumulation leads to cell death. How much accumulates depends on the pace of kynureninase. observed in studies Source 3, 1
   ⚖ When the balance tips

   too much — When KMO runs faster than the kynureninase after it, 3-hydroxykynurenine backs up — the compound that releases electrons and forms radicals with iron and copper. In cell culture, accumulation leads to cell death.

   too little — When this step lags, more kynurenine goes to kynurenic acid, which damps at the receptor instead of exciting it.

   observed in studies · Source 3, 1

   Field of research — The build-up of this compound is studied in disorders of the nervous system. Source 9, 3

3. 3-Hydroxykynurenine → 3-OH-anthranilic acid Kynureninase · Vitamin B6 (PLP) Kynureninase cleaves the side chain from 3-hydroxykynurenine, forming 3-hydroxyanthranilic acid. This enzyme also works with pyridoxal phosphate, the active form of vitamin B6. If little vitamin B6 is available, the steps before it back up. Source 1↕ both, depending on amount This compound releases electrons and forms radicals with iron and copper; in cell culture it shows both sides, depending on its surroundings. Its turnover hangs on vitamin B6 — when little is around, earlier stages back up and more goes into the side routes. observed in studies Source 1
   ⚖ When the balance tips

   too much — When this compound backs up, it too forms radicals with iron and copper; in cell culture it shows both sides, depending on its surroundings.

   too little — When little vitamin B6 is around, kynureninase cannot keep up. More material then diverts into xanthurenic acid, which the body excretes.

   observed in studies · Source 1

4. 3-OH-anthranilic acid → Quinolinic acid 3-HAO · Iron An iron-containing enzyme opens the ring; the intermediate ACMS rearranges spontaneously into quinolinic acid. In the nervous system, quinolinic acid binds to NMDA receptors. At the receptor it has an excitatory action. Source 1↕ both, depending on amount As a building block, quinolinic acid is the way forward: QPRT converts it to NAD⁺. At the NMDA receptor it acts excitatory and lets calcium in; if the channel stays open too long, this burdens the nerve cell. Which side prevails depends on the pace of QPRT. observed in studies Source 1
   ⚖ When the balance tips

   too much — When quinolinic acid backs up, more of it stays at the NMDA receptor. In cell culture and animal models this holds the calcium channel open longer than the cell buffers the load.

   too little — When little arrives, NAD⁺ has to come from niacin; the long route from tryptophan then contributes little.

   observed in studies · Source 1

   Field of research — Its action at the NMDA receptor is studied in disorders of the nervous system. Source 9, 1

5. Quinolinic acid → NAD⁺ QPRT · PRPP Both routes end here: QPRT converts quinolinic acid to NAD⁺ over two steps, while NAPRT and NAMPT convert nicotinic acid and nicotinamide. Tryptophan thus supplies NAD⁺ alongside the shorter route from niacin (vitamin B3). Many enzymes use it up constantly. Source 4, 1, 6↑ supplies NAD⁺ accepts hydrogen and passes it on; without this carrier, glycolysis, the citrate cycle and fatty acid breakdown do not run. NAD⁺ is also consumed: sirtuins, PARP and CD38 cleave it. Energy production and DNA repair share the same pool. established physiology Source 4, 6
   ⚖ When the balance tips

   too much — The pool does not pile up: the cell makes NAD⁺ as needed, and sirtuins, PARP and CD38 consume it continuously. What rises is turnover, not a heap.

   too little — When the pool falls, energy production and DNA repair reach for the same molecules — both draw from one pot.

   established physiology · Source 4, 6

6. Niacin (vitamin B3) → NAD⁺ NAPRT, NAMPT · PRPP Both routes end here: QPRT converts quinolinic acid to NAD⁺ over two steps, while NAPRT and NAMPT convert nicotinic acid and nicotinamide. Tryptophan thus supplies NAD⁺ alongside the shorter route from niacin (vitamin B3). Many enzymes use it up constantly. Source 4, 1, 6↑ supplies NAD⁺ accepts hydrogen and passes it on; without this carrier, glycolysis, the citrate cycle and fatty acid breakdown do not run. NAD⁺ is also consumed: sirtuins, PARP and CD38 cleave it. Energy production and DNA repair share the same pool. established physiology Source 4, 6
   ⚖ When the balance tips

   too much — The pool does not pile up: the cell makes NAD⁺ as needed, and sirtuins, PARP and CD38 consume it continuously. What rises is turnover, not a heap.

   too little — When the pool falls, energy production and DNA repair reach for the same molecules — both draw from one pot.

   established physiology · Source 4, 6

Further stations

• Tryptophan — amino acid from food Tryptophan is an amino acid the body cannot make itself. Some is built into proteins and a small part is converted to serotonin; the largest part enters the kynurenine pathway. The routes share one pool. Source 1↑ supplies Tryptophan feeds several routes at once: protein building, serotonin, this pathway and – via gut bacteria – the indoles. All draw on the same pool — if one is used more, less is left for the others. A change in one route therefore acts back on the others. established physiology Source 1
  ⚖ When the balance tips

  too much — More tryptophan in the blood does not mean more serotonin: at the blood-brain barrier it shares its transporter with other large amino acids, and the hydroxylase is the slowest step. The surplus runs into the kynurenine pathway.

  too little — When little tryptophan arrives, all routes share the same smaller pool. Protein building comes first; the metabolic routes get what is left.

  established physiology · Source 1

• Kynurenic acid — side product Kynurenine aminotransferases (KAT) convert kynurenine to kynurenic acid; they work with vitamin B6. Kynurenic acid binds to glutamate receptors and is excreted in the urine. At the NMDA receptor it acts opposite to quinolinic acid. Source 5, 1↕ both, depending on amount Kynurenic acid occupies binding sites for glutamate and damps excitation there — at the NMDA receptor it thus acts opposite to quinolinic acid. The same damping that curbs over-excitation also lowers ordinary signal transmission. observed in studies Source 5, 1
  ⚖ When the balance tips

  too much — More kynurenic acid occupies more binding sites for glutamate. The same damping that curbs over-excitation also lowers ordinary signal transmission.

  too little — When the KAT enzymes run slower — for instance when little vitamin B6 is around — the counterpart to quinolinic acid at the same receptor is missing.

  observed in studies · Source 5, 1

  Field of research — The ratio of kynurenic acid to quinolinic acid is studied in disorders of the nervous system. Source 9, 5

• Xanthurenic acid — from 3-hydroxykynurenine The same KAT enzymes also convert 3-hydroxykynurenine; this produces xanthurenic acid, which is excreted in the urine. This branch removes material from the pathway. Source 5, 1↓ depletes This branch carries material out of the pathway: xanthurenic acid is lost in the urine instead of being passed on to NAD⁺. It is used more when little vitamin B6 is around — kynureninase gives up the cofactor earlier than the KAT enzymes do. established physiology Source 5, 1
  ⚖ When the balance tips

  too much — When the amount rises, the route before it is stalling: kynureninase gives up its cofactor vitamin B6 earlier than the KAT enzymes do, and 3-hydroxykynurenine diverts into this branch.

  too little — Little xanthurenic acid means the main route is running through and hardly any material diverts sideways.

  established physiology · Source 5, 1

• Picolinic acid — branch via ACMS Part of the intermediate ACMS is converted by the enzyme ACMSD into picolinic acid instead of rearranging into quinolinic acid. How much follows this branch depends on the amount of this enzyme in the tissue. What branches off here adds no NAD⁺. Source 1↓ depletes This branch removes material from the main route: what ACMSD converts to picolinic acid never reaches quinolinic acid and contributes no NAD⁺. Picolinic acid binds metals such as zinc and iron and is excreted in the urine. established physiology Source 1
  ⚖ When the balance tips

  too much — The more ACMSD converts, the less ACMS rearranges into quinolinic acid — the branch takes material from the main route before NAD⁺ forms.

  too little — When ACMSD works little, almost all ACMS rearranges into quinolinic acid by itself, and the main route carries the whole throughput.

  established physiology · Source 1

• Niacin (vitamin B3) — nicotinic acid, nicotinamide Niacin is the collective name for nicotinic acid and nicotinamide from food. NAPRT converts nicotinic acid and NAMPT converts nicotinamide; both routes reach the same intermediates as the tryptophan route and end at NAD⁺. Niacin spares the long route. Source 6, 4↑ supplies Niacin saves the body the long route: once absorbed from food, it reaches NAD⁺ in a few steps, while the route from tryptophan runs over many stages. The more niacin arrives, the less NAD⁺ has to be made from tryptophan. established physiology Source 6, 4
  ⚖ When the balance tips

  too much — When plenty of niacin arrives from food, the short route covers the need for NAD⁺, and the long route from tryptophan matters less.

  too little — When little arrives, NAD⁺ hangs more on tryptophan — and thereby on every step before it, including the cofactors iron and vitamin B6.

  established physiology · Source 6, 4

Cofactors in this pathway

• Vitamin B6 — As pyridoxal phosphate, cofactor of kynureninase and the kynurenine aminotransferases; with little PLP, steps back up Source 1, 5In the ORY catalogue as a laboratory value: Vitamin B6
• Iron — Haem iron in IDO and TDO; another iron enzyme opens the ring of 3-hydroxyanthranilic acid; without it the pathway stalls Source 2, 1In the ORY catalogue as a laboratory value: Eisen
• Niacin (vitamin B3) — Nicotinic acid and nicotinamide are precursors from which cells build NAD⁺ even without this route; the shorter route Source 6
• Vitamin B2 (FAD) — As FAD, cofactor of kynurenine 3-monooxygenase (KMO); without FAD, KMO adds no OH group Source 3
• NADPH — Supplies KMO with the electrons for adding the OH group; it is used up in the process Source 3
• PRPP — Supplies QPRT with the sugar-phosphate part on the way to NAD⁺; without PRPP, quinolinic acid yields no NAD⁺ Source 4

What acts on this pathway

• Inflammatory signals — Interferon-γ from immune cells, TNF and bacterial LPS make macrophages, dendritic cells and vessel-wall cells read the IDO gene more strongly. More tryptophan then enters this route, and the kynurenine-to-tryptophan ratio rises. Source 7, 2
• Cortisol — In the liver the related enzyme TDO carries out the first step. Cortisol and related glucocorticoids make the liver cell form more TDO; more tryptophan then runs along the kynurenine pathway there. Source 1
• Inflammation at KMO — The same signals — interferon-γ, TNF and bacterial LPS — also make immune cells build more KMO. More kynurenine then goes towards 3-hydroxykynurenine and quinolinic acid, and less into kynurenic acid. Source 7, 3

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. Stimulus at gut wall → CXCR3 receptor Gliadin fragments bind to the CXCR3 receptor of the intestinal cell. The signal is passed inwards via the signalling protein MyD88. Without CXCR3 and MyD88, release does not occur. Source 1↑ supplies CXCR3 is the entry gate for gliadin: only binding there sets off the release of zonulin via MyD88. In cell culture and animal models, release does not occur without CXCR3 or MyD88. observed in studies Source 1
   ⚖ When the balance tips

   too much — If intestinal cells carry more CXCR3 on their surface, more gliadin fragments bind, and the signal to release zonulin is stronger.

   too little — If CXCR3 or MyD88 is missing, the signal does not reach the inside of the cell; in animal models the intestinal cells then release hardly any zonulin in response to gliadin.

   observed in studies · Source 1

2. CXCR3 receptor → Zonulin is released · MyD88 In response, the cell releases zonulin to the outside. Zonulin is the precursor of the protein haptoglobin 2, which is why it is also called pre-haptoglobin 2. From outside it acts on the junctions between the cells. Source 2, 1↑ supplies Zonulin is the link between stimulus and barrier: once released, it acts back on the same and neighbouring cells. Only the uncleaved form loosens the junctions; once cleaved, it loses this action. observed in studies Source 2, 1
   ⚖ When the balance tips

   too much — If a lot of zonulin is released, more receptors on the cell surface are occupied, and the junctions between the cells stay loosened for longer.

   too little — If little zonulin is released, the signal to loosen does not come, and the junctions between the cells stay largely closed.

   observed in studies · Source 1, 3

3. Zonulin is released → Zonulin in stool Part of the released zonulin is found in stool and in blood. Studies show that widely used test kits do not capture pre-haptoglobin 2 itself, but related proteins such as properdin. Source 5
4. PAR2 and EGFR receptors → Signal inside the cell Protein kinase C The receptor switches on protein kinase C. It attaches phosphate groups to the proteins of the junctions between two intestinal cells. This loosens the junctions. Source 1↕ both, depending on amount Protein kinase C alters the proteins of the junction by attaching phosphate groups. Depending on the type of kinase and the site of the phosphate group, the junction is loosened or built more tightly. observed in studies Source 1, 7
   ⚖ When the balance tips

   too much — If protein kinase C stays active for long, more phosphate groups remain on the junction proteins, and the actin scaffold stays contracted.

   too little — If the kinase is barely active, for example because little diacylglycerol and calcium are present, the junction proteins stay unchanged, and the zonulin signal does not reach the junction.

   observed in studies · Source 1, 6

5. Signal inside the cell → Tight junction opens · actin scaffold The junctions loosen and the actin scaffold contracts. As a result, the gap between two intestinal cells widens for a short time. If the pull persists, the gap stays open. Source 4, 1↕ both, depending on amount When the actin scaffold contracts, it pulls on the sealing proteins and widens the gap. Opened briefly, this is a reversible process; if the pull persists, the gap stays open too. observed in studies Source 1, 4
   ⚖ When the balance tips

   too much — If the seam stays loosened for long, more components of the gut contents cross into the gut wall and meet immune cells there.

   too little — If the seam barely opens, the route between the cells stays narrow; water and ions then pass mainly through the cells themselves via channels and pumps.

   observed in studies · Source 1, 4

6. Tight junction opens → Path between the cells Water, ions and small molecules pass between the cells along this route. When the phosphate groups are removed again, the gap closes. Open for long, it also lets larger molecules into the gut wall. Source 1, 3↕ both, depending on amount Water with ions passes along the opened route into the gut lumen and carries bacteria away. If it stays open for long, larger molecules such as food and bacterial components also reach the gut wall. observed in studies Source 1, 3
   ⚖ When the balance tips

   too much — If the route stays open for long, more food and bacterial components get beneath the cell layer; there they trigger a response from immune cells.

   too little — If the route is barely open, only little water and few ions pass between the cells, and the outflow into the gut lumen stays small.

   observed in studies · Source 1, 3

Further stations

• Stimulus at gut wall — bacteria, gliadin fragments Certain bacteria in the small intestine and fragments of the wheat protein gliadin reach the surface of the intestinal cells. They respond by releasing zonulin. Source 1↑ supplies Bacteria and gliadin are the two described triggers to which intestinal cells respond by releasing zonulin. The water flow into the gut lumen that follows carries bacteria away. observed in studies Source 1
  ⚖ When the balance tips

  too much — If gliadin fragments or bacteria keep reaching the gut wall, the cells release zonulin continuously, and the junctions between them stay loosened for longer.

  too little — If few such stimuli arrive, the intestinal cells release hardly any zonulin, and the junctions between them stay largely closed.

  observed in studies · Source 1

• PAR2 and EGFR receptors — on the cell surface From the outside, zonulin binds to the PAR2 and EGFR receptors of the same or neighbouring cells. This is where its action at the barrier begins. EGFR is switched on via PAR2 as well. Source 3, 1↑ supplies When zonulin binds to PAR2, EGFR is switched on at the same time via this receptor; both pass the signal to loosen into the cell. In cell culture and animal models, the action does not occur without PAR2. observed in studies Source 2, 3
  ⚖ When the balance tips

  too much — If many receptors are occupied by zonulin, the signal into the cell is triggered more often, and the junctions loosen further.

  too little — If the receptors are barely occupied or PAR2 is missing, the signal does not come; in cell culture and animal models the barrier then stays tight.

  observed in studies · Source 2, 3

• ZO-1, occludin, claudins — junction proteins The junction consists of several proteins: claudins and occludin form the seal, and ZO-1 anchors it to the actin scaffold. The claudins determine how tight the seam is. Source 4↑ supplies Claudins determine which ions and how much water pass through the gap; occludin and ZO-1 hold the seal to the actin scaffold. Depending on the type of claudin, the seam is tighter or more permeable. established physiology Source 4, 7
  ⚖ When the balance tips

  too much — If many sealing claudins are built in, the gap narrows, and fewer ions and less water pass between the cells.

  too little — If few junction proteins are built in or ZO-1 is not anchored to actin, no tight seal forms, and more substances pass between the cells.

  established physiology · Source 4

Cofactors in this pathway

• Calcium — Messenger inside the cell; together with protein kinase C it sets how the junctions are assembled Source 7, 1In the ORY catalogue as a laboratory value: Calcium (intrazellulär)
• Diacylglycerol — Lipid messenger that frees protein kinase C from its own inhibition and so sets off the loosening of the junctions Source 6
• ATP — Phosphate donor of protein kinase C; it also sits bound within the actin scaffold; without ATP no phosphorylation Source 6, 8
• Magnesium — Binds together with ATP inside actin, the scaffold beneath the junctions; needed to build actin filaments Source 8In the ORY catalogue as a laboratory value: Magnesium

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. Histidine → Histamine Histidine decarbox. · vitamin B6 (PLP) The enzyme histidine decarboxylase removes a carboxyl group from histidine; this produces histamine. The enzyme needs pyridoxal phosphate, the active form of vitamin B6. Histamine acts on vessels, stomach, nerves and immune cells. Source 2, 1↕ both, depending on amount Histamine is a messenger with many tasks: it widens blood vessels, stimulates stomach acid, acts on wakefulness in the brain and steers immune cells. How it acts depends on amount, site and receptor. established physiology Source 3, 4
   ⚖ When the balance tips

   too much — If more histamine forms or arrives than DAO and HNMT break down, it builds up and acts at more receptors at once: vessels widen, gut movement increases, skin and mucous membranes react.

   too little — If little histamine is available, for example when histidine decarboxylase is absent, its signals are weaker; in animal models without this enzyme, immune cells respond differently.

   observed in studies · Source 4, 2

2. Histamine → Storage granules · heparin Mast cells and basophil blood cells store histamine in small vesicles. There it stays bound to heparin and has no effect on the outside. In this way the cell keeps a store ready. Source 3↑ supplies In the granules, histamine is bound to heparin and inactive. In this way the mast cell keeps a store ready that it can release within seconds when activated. established physiology Source 3
   ⚖ When the balance tips

   too much — If many mast cells with filled granules are present, a larger amount of histamine can be released at once on activation.

   too little — If the granules are emptied after a release, the cell needs time to form and store new histamine; until then it releases less.

   observed in studies · Source 3

3. Storage granules → Release When an antigen binds to IgE antibodies on the mast cell, or other stimuli act on it, the vesicles fuse with the cell membrane and release histamine to the outside. Only now can histamine act. Source 3↑ supplies Release brings histamine to the receptors nearby; together with it, mast cells release further substances such as tryptase and heparin. Only now does stored histamine become active. established physiology Source 3
   ⚖ When the balance tips

   too much — If many mast cells are activated at once, histamine also enters the blood and acts on vessels throughout the body: they widen and let more fluid into the tissue.

   too little — If little is released, histamine stays bound in the granules, and its action is confined to the area around the cell or does not occur.

   established physiology · Source 3, 5

4. Release → H1 to H4 receptors Histamine binds to four receptor types. Depending on type and tissue, they trigger different signals – on blood vessels, nerve cells, stomach cells and immune cells. H1 widens vessels, for example, and H2 stimulates stomach acid. Source 3, 5↕ both, depending on amount H1 widens vessels, makes them more permeable and signals itch, H2 stimulates stomach acid, H3 slows the release of messengers in the nervous system, H4 steers immune cells. established physiology Source 3, 5
   ⚖ When the balance tips

   too much — If a lot of histamine is bound to H1, vessels widen, fluid passes into the tissue and nerve endings signal itch; at H2 more stomach acid is formed.

   too little — If the receptors are little occupied or blocked, for example by H1 antihistamines, these signals do not occur; in the brain, blocking H1 causes drowsiness.

   established physiology · Source 5, 3

5. Free histamine → Imidazole acetic acid DAO · copper Diamine oxidase removes the amino group. Via an aldehyde intermediate, imidazole acetic acid is formed and excreted in the urine. This ends the action of this histamine. Source 4↓ depletes With this step, histamine outside the cells has been broken down. Imidazole acetic acid leaves the body in the urine. established physiology Source 4
   ⚖ When the balance tips

   too much — If a lot of histamine is broken down via DAO, more imidazole acetic acid forms; its amount follows turnover outside the cells.

   too little — If DAO works more slowly, less imidazole acetic acid forms, and more histamine stays free or is methylated inside the cell instead.

   established physiology · Source 4

6. N-methylhistamine → N-MIAA MAO-B, ALDH Monoamine oxidase B and an aldehyde dehydrogenase break N-methylhistamine down further. The end product appears in the urine. In this way methylated histamine leaves the body. Source 4↓ depletes N-methylimidazole acetic acid is the end product of breakdown inside the cell. Through it, methylated histamine leaves the body in the urine. established physiology Source 4, 6
   ⚖ When the balance tips

   too much — If more histamine is formed and methylated, more N-MIAA appears in the urine; the amount follows turnover via the route inside the cell.

   too little — If MAO-B is inhibited or little FAD is available, more N-methylhistamine remains unchanged and appears as such in the urine.

   established physiology · Source 4, 6

Further stations

• Histidine — amino acid from food Histidine is an amino acid. It comes from dietary protein and from the turnover of the body's own proteins, and it is the starting material for histamine. Only a small part of it becomes histamine. Source 1↑ supplies Histidine is a building block of proteins and the starting material for histamine, carnosine and other substances. Only a small part of histidine goes into histamine formation. established physiology Source 1
  ⚖ When the balance tips

  too much — If a lot of histidine arrives, the liver breaks the excess down via urocanic acid to glutamate. How much histamine forms depends mainly on the amount of histidine decarboxylase.

  too little — If little histidine is available, the body draws on stores, for example carnosine in the muscles and haemoglobin.

  established physiology · Source 1, 2

• DAO — diamine oxidase, with copper Diamine oxidase is a copper-containing enzyme. Cells of the intestinal lining, the kidney and the placenta release it to the outside; it works outside the cells. It intercepts histamine from food in the gut. Source 4↓ depletes Diamine oxidase breaks histamine down outside the cells, above all in the gut. There it intercepts histamine from food before it reaches the blood. established physiology Source 4
  ⚖ When the balance tips

  too much — If a lot of DAO is active, free histamine in the gut is broken down quickly and little of it reaches the blood; in pregnancy the placenta forms particularly large amounts of DAO.

  too little — If little DAO is active, for example when medicines or alcohol inhibit it or copper is scarce, histamine from food is broken down more slowly and passes more readily into the blood.

  established physiology · Source 4

• Free histamine — outside the cells Outside the cells, histamine is present in free form. In the gut, histamine is added from foods and from bacteria that convert histidine; part of it can be detected in stool. It acts at receptors until DAO breaks it down. Source 4↕ both, depending on amount Free histamine acts at receptors in the gut wall and nearby until DAO breaks it down. Histamine from foods and from bacteria adds to the body's own amount. established physiology Source 4
  ⚖ When the balance tips

  too much — If more histamine arrives with food than DAO can break down, part of it passes through the gut wall into the blood and acts on vessels, skin and gut.

  too little — If little free histamine is present, the receptors nearby remain largely unoccupied; DAO then finds little substrate.

  established physiology · Source 4

• N-methylhistamine — formed inside the cell Cells absorb histamine. Inside, histamine N-methyltransferase transfers a methyl group from SAM to the molecule; this produces N-methylhistamine. This ends its action inside the cell. Source 4↓ depletes Methylation by HNMT ends the action of histamine inside the cell. In the brain and the airways, where there is hardly any DAO, it is the main breakdown route. established physiology Source 4
  ⚖ When the balance tips

  too much — If a lot of histamine is methylated inside the cell, more N-methylhistamine forms and more SAM is used up.

  too little — If little SAM is available or HNMT works more slowly, histamine inside the cell is broken down more slowly and acts there for longer.

  established physiology · Source 4

Cofactors in this pathway

• Histidine — Starting material from which histidine decarboxylase makes histamine; only a small part becomes histamine Source 1, 2In the ORY catalogue as a laboratory value: Histidin
• Vitamin B6 — Cofactor (PLP) of histidine decarboxylase; without PLP no histamine forms Source 2In the ORY catalogue as a laboratory value: Vitamin B6
• Copper — Metal in the active site of diamine oxidase, which breaks histamine down outside cells; without copper no DAO activity Source 4In the ORY catalogue as a laboratory value: Kupfer (Cu)
• SAM (from methionine) — Donor of the methyl group for histamine N-methyltransferase inside the cell; with little of it, this breakdown stalls Source 4In the ORY catalogue as a laboratory value: Methionin
• Vitamin B2 (FAD) — Flavin cofactor of monoamine oxidase B, which breaks N-methylhistamine down further; without FAD this step stalls Source 6, 4

What acts on this pathway

• H1 antihistamines — Drugs in this group occupy the H1 receptor and hold it in its inactive form. Histamine can then no longer bind there; the other receptor types are not affected. Source 5

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. Homocysteine → Cystathionine CBS · vitamin B6 (PLP), serine Cystathionine β-synthase (CBS) joins homocysteine to serine. The enzyme works with pyridoxal phosphate, the active form of vitamin B6, and also carries a haem group. From here the sulphur no longer returns to methionine. Source 2↑ supplies Cystathionine holds the sulphur of homocysteine until CSE releases cysteine from it. Once it is formed, the route is fixed: this sulphur no longer returns to methionine. established physiology Source 2
   ⚖ When the balance tips

   too much — If cystathionine builds up because CSE works more slowly, more of it passes into the urine, and less cysteine is formed.

   too little — If little cystathionine forms, for example because little PLP is available for CBS, less sulphur runs towards cysteine, and homocysteine stays longer in the methionine cycle.

   established physiology · Source 2

2. Cystathionine → Cysteine CSE · vitamin B6 (PLP) Cystathionine γ-lyase (CSE) splits cystathionine, releasing cysteine. Cysteine also comes from food and is the building block in shortest supply for glutathione production. When little cysteine is available, less glutathione forms. Source 2, 1↑ supplies Cysteine supplies the sulphur group that makes glutathione active. Because it is usually the scarcest building block, its supply, together with GCL, determines how much glutathione the cell can make. established physiology Source 1
   ⚖ When the balance tips

   too much — If more cysteine is present than glutathione synthesis needs, the excess is broken down, among other things to taurine, sulphate and hydrogen sulphide; outside the cell cysteine quickly oxidises to cystine.

   too little — If little cysteine is available, the cell makes less glutathione, even when glutamate, glycine and ATP are plentiful.

   established physiology · Source 1, 2

3. Cysteine → γ-glutamylcysteine GCL · glutamate, ATP Glutamate-cysteine ligase (GCL) links glutamate and cysteine through an unusual bond. This step sets the pace of the whole pathway and uses ATP. Finished glutathione slows GCL by feedback. Source 1↑ supplies GCL sets the pace of glutathione synthesis. Finished glutathione inhibits it by feedback; when the glutathione pool falls, the inhibition eases and this step runs faster. established physiology Source 1
   ⚖ When the balance tips

   too much — If γ-glutamylcysteine builds up because glutathione synthetase cannot keep pace, it is converted to 5-oxoproline, which is excreted in the urine.

   too little — If little γ-glutamylcysteine forms because cysteine or ATP is scarce, the downstream synthetase makes correspondingly less glutathione; it is itself rarely the bottleneck.

   established physiology · Source 1

4. γ-glutamylcysteine → Glutathione (GSH) Glutathione synthetase · glycine, ATP Glutathione synthetase adds glycine. The result is glutathione – a small molecule made of glutamate, cysteine and glycine that is present in every cell. The liver also releases it into the blood. Source 1↑ supplies Glutathione supplies electrons for the peroxidases and the binding partner for the transferases. The liver makes a particularly large amount and releases it into the blood; other tissues recover cysteine from it. established physiology Source 1
   ⚖ When the balance tips

   too much — If a lot of glutathione is present, it inhibits GCL and so slows its own synthesis; the cell thereby keeps its pool within a narrow range.

   too little — If little glutathione is available, the feedback inhibition of GCL eases, and via Nrf2 the cell makes more enzymes of glutathione synthesis; until then peroxides and foreign compounds are processed more slowly.

   established physiology · Source 1

5. Hydrogen peroxide → Water GPx · selenium Glutathione peroxidases transfer hydrogen from two GSH molecules to hydrogen peroxide, which becomes water. These enzymes carry selenium in their active site. Two GSH are used up per molecule of peroxide. Source 3↓ depletes With the formation of water the peroxide is defused. For this, glutathione peroxidase uses up two molecules of GSH per molecule of hydrogen peroxide. established physiology Source 3
   ⚖ When the balance tips

   too much — If a lot of peroxide passes through this reaction, a correspondingly large amount of GSH becomes GSSG; glutathione reductase must then use more NADPH to recover it.

   too little — If the reaction runs slowly, for example because little selenium is available for the peroxidase, hydrogen peroxide persists longer; enzymes such as catalase and peroxiredoxins then handle part of it.

   established physiology · Source 3, 4

6. GSH → GSSG In this reaction, two GSH molecules join via their sulphur atoms to form GSSG, glutathione disulphide. If GSSG builds up, the cell exports it and so loses glutathione. Source 3, 4↓ depletes GSSG is spent glutathione. If it accumulates, it can attach glutathione to sulphur groups of proteins and alter their activity; many cells also export it. established physiology Source 4, 1
   ⚖ When the balance tips

   too much — If GSSG builds up because the reductase cannot keep pace, the cell exports it and thereby loses glutathione; at the same time more proteins carry glutathione as a tag.

   too little — If little GSSG forms, little peroxide is currently being broken down via this route; the ratio then lies far on the side of GSH.

   established physiology · Source 4, 1

7. GSSG → GSH regenerated Glutathione reductase · NADPH, FAD (vitamin B2) Glutathione reductase splits GSSG back into two GSH. It does this by transferring hydrogen from NADPH and carries FAD, which is derived from vitamin B2. So not every molecule has to be made anew. Source 4↑ supplies The reductase recovers GSH without new glutathione having to be made. It needs NADPH for this, which comes mainly from the pentose phosphate pathway; the cycle thus depends on sugar metabolism. established physiology Source 4
   ⚖ When the balance tips

   too much — If the reductase recovers plenty of GSH, almost all glutathione is in the reduced form, and hardly any GSSG is exported from the cell.

   too little — If little GSH is recovered because NADPH or FAD is scarce, GSSG accumulates, and the cell has to replace glutathione through slower new synthesis.

   established physiology · Source 4, 1

8. Foreign compound → GSH conjugate GST · GSH Glutathione S-transferases (GST) link glutathione to the foreign compound. The product is more water-soluble than the starting substance and is exported from the cell. For a few substances the product is more reactive than the starting substance. Source 5↕ both, depending on amount Usually the conjugate is less reactive than the starting substance and is moved out of the cell by transporters. With some substances, such as dihaloalkanes, a more reactive product forms instead. established physiology Source 5
   ⚖ When the balance tips

   too much — If conjugates accumulate in the cell because export cannot keep pace, they inhibit the glutathione S-transferases; the pathway then slows itself.

   too little — If little conjugate forms because little glutathione or little transferase activity is available, the foreign compound remains reactive longer and forms other bonds.

   observed in studies · Source 5

9. GSH conjugate → Mercapturic acid Peptidases, NAT · acetyl-CoA Peptidases trim the conjugate down to the cysteine residue; an acetyl group is added. As mercapturic acid, the substance leaves the body in the urine. A cysteine residue leaves the body with it. Source 5, 4↓ depletes With mercapturic acid the foreign compound leaves the body. The glutamate and glycine of glutathione are split off beforehand and reused; the cysteine residue leaves with it. established physiology Source 5
   ⚖ When the balance tips

   too much — If a lot of mercapturic acid is excreted, a cysteine residue leaves the body with each molecule; this sulphur must be replenished from food or transsulfuration.

   too little — If little mercapturic acid forms, little foreign compound is currently being bound via this route, and the cysteine residue stays in the body.

   established physiology · Source 5, 2

Further stations

• Homocysteine — from methionine conversion Homocysteine arises when the amino acid methionine is converted. Part of it enters the transsulfuration pathway, which leads to cysteine. When plenty of S-adenosylmethionine is present, more of it goes to cysteine. Source 2↑ supplies Homocysteine supplies the sulphur for cysteine via the transsulfuration pathway. Whether it goes there or back to methionine is regulated by S-adenosylmethionine: when plenty of it is present, it switches on CBS. established physiology Source 2
  ⚖ When the balance tips

  too much — If homocysteine builds up because neither the path to cysteine nor the one back to methionine keeps pace, more of it passes from the cells into the blood.

  too little — If little homocysteine is available, less sulphur flows into the transsulfuration pathway; the cysteine for glutathione must then come more from food.

  established physiology · Source 2, 1

• GSH — reduced form In the cell, glutathione is mostly present in its reduced form. The SH group of cysteine can give up hydrogen; the following reactions are based on this. It thus keeps the cell interior in a reduced state. Source 4↑ supplies Reduced glutathione gives electrons to peroxidases and to proteins whose sulphur groups have been oxidised. It thus keeps the cell interior reduced; how strongly depends on the ratio of GSH to GSSG. established physiology Source 4
  ⚖ When the balance tips

  too much — If GSH strongly outweighs GSSG, glutaredoxins quickly remove glutathione tags from proteins again; switching events via oxidised sulphur groups then occur less often.

  too little — If little GSH is available, the ratio shifts towards GSSG; more sulphur groups in proteins stay oxidised or carry glutathione as a tag, and enzymes with such groups change their activity.

  observed in studies · Source 4

• Hydrogen peroxide — by-product of metabolism Hydrogen peroxide is formed continuously when cells use oxygen. If it is not converted, it can alter fatty acids and proteins. In small amounts it also serves the cell as a messenger. Source 3↕ both, depending on amount In small amounts hydrogen peroxide acts as a messenger that switches enzymes on and off via their sulphur groups; in larger amounts it oxidises fatty acids and proteins. The peroxidases determine how long it remains. observed in studies Source 3
  ⚖ When the balance tips

  too much — If hydrogen peroxide builds up, it oxidises sulphur groups in proteins and fatty acids in membranes; at the same time more GSH is used up and turned into GSSG.

  too little — If there is very little hydrogen peroxide, signals are missing by which cells respond, for example, to growth factors and insulin; a cell is never entirely free of it.

  observed in studies · Source 3

• Foreign compound — xenobiotic molecule Many substances foreign to the body, and their breakdown products, have a site to which sulphur can attach. Glutathione intercepts such substances and is used up in the process. Source 5↓ depletes Electrophilic foreign compounds, i.e. substances that pull electrons towards themselves, react with sulphur groups of proteins and with DNA. Glutathione intercepts them and is itself used up in the process. established physiology Source 5
  ⚖ When the balance tips

  too much — If a large amount of such a compound arrives at once, glutathione is used up faster than the cell can make it; the remaining compound then binds to the cell's proteins.

  too little — If little of it arrives, cells make the transferases at their basal level; only reactive compounds switch on the production of further transferases via Nrf2.

  established physiology · Source 5, 6

Cofactors in this pathway

• Cysteine — Building block of glutathione and the part that runs short first; with little of it, less glutathione forms Source 1, 2
• Glutamate — Building block of glutathione; GCL joins it to cysteine; this step sets the pace Source 1In the ORY catalogue as a laboratory value: Glutamat
• Glycine — Building block of glutathione; glutathione synthase adds it last; without glycine no finished glutathione Source 1In the ORY catalogue as a laboratory value: Glycin
• Vitamin B6 — Cofactor (PLP) of CBS and CSE on the way from homocysteine to cysteine; with little PLP, less cysteine forms Source 2In the ORY catalogue as a laboratory value: Vitamin B6
• Selenium — Present as selenocysteine in the active site of the glutathione peroxidases; when scarce, peroxide breakdown slows Source 3In the ORY catalogue as a laboratory value: Selen
• NADPH — Hydrogen donor of glutathione reductase, which turns GSSG back into two GSH; when scarce, GSSG builds up Source 4
• Vitamin B2 (FAD) — Flavin cofactor of glutathione reductase; with little FAD, less GSH is regenerated Source 4
• Serine — Partner of homocysteine in the CBS reaction that leads to cystathionine; without serine no cystathionine forms Source 2In the ORY catalogue as a laboratory value: Serin

What acts on this pathway

• Paracetamol — When paracetamol is broken down in the liver, the reactive intermediate NAPQI is formed. Glutathione S-transferases bind it to glutathione for excretion; glutathione is used up in the process. Source 6

The pathway step by step

Each station states what the compound does there. Three signs: ↑ supplies — builds up or makes available · ↓ depletes — inhibits, consumes or withholds · ↕ both, depending on amount. Behind it stands what the statement rests on: established physiology, observed in studies, or contested. The signs do not grade; they name the direction.

1. L-arginine → Nitric oxide NO synthase · BH4, haem, NADPH, O₂ NO synthases convert arginine with oxygen into nitric oxide and citrulline. They need NADPH, haem iron and the cofactor tetrahydrobiopterin (BH4). NO acts as a signalling molecule, for example in blood vessels and nerves. NO and superoxide form peroxynitrite. Source 1, 2↕ both, depending on amount In small amounts NO is a signalling molecule in blood vessels and nerves. If it meets superoxide, it reacts with it to form peroxynitrite; how much NO and superoxide meet decides which route prevails. established physiology Source 1, 3
   ⚖ When the balance tips

   too much — If cells make a lot of NO while superoxide is present, more peroxynitrite forms, and more 3-nitrotyrosine is left on proteins.

   too little — If little NO forms, less signal reaches blood vessels and nerves; superoxide is then mostly converted by the superoxide dismutases instead of being captured by NO.

   observed in studies · Source 3

2. Nitric oxide → Peroxynitrite · Superoxide (O₂•⁻) When NO meets the superoxide radical, the two combine to form peroxynitrite at almost every encounter. Superoxide arises in mitochondria and from the NADPH oxidases of immune cells. It oxidises sulphur groups and metal centres. Source 3, 7↓ depletes Peroxynitrite is a strong oxidant: it oxidises sulphur groups in proteins and reacts with metal centres. Via carbon dioxide it releases radicals that nitrate tyrosine. established physiology Source 3, 4
   ⚖ When the balance tips

   too much — If more peroxynitrite forms than peroxiredoxins and glutathione peroxidases can capture, more radicals reach proteins and fats; in cell culture this disturbs the work of the mitochondria.

   too little — If little peroxynitrite forms, hardly any nitration occurs on proteins; immune cells then also use less of this oxidant against microorganisms.

   observed in studies · Source 3, 5

3. Peroxynitrite → Radical pair · Carbon dioxide (CO₂) In the body, peroxynitrite usually reacts first with carbon dioxide. The resulting intermediate splits into two radicals: nitrogen dioxide and the carbonate radical. Both alter proteins close by. Source 3, 4↓ depletes Nitrogen dioxide and the carbonate radical are short-lived and react close by: they remove electrons from tyrosine and other building blocks and so alter proteins. established physiology Source 3, 4
   ⚖ When the balance tips

   too much — If many radical pairs form, more tyrosine residues are nitrated; some of the radicals are captured beforehand by glutathione and other antioxidants.

   too little — If little carbon dioxide is at hand, fewer radical pairs form; peroxynitrite then reacts more often directly with sulphur groups and metal centres.

   observed in studies · Source 3, 4

4. Radical pair → 3-Nitrotyrosine · Tyrosine in protein The carbonate radical removes an electron from the amino acid tyrosine; nitrogen dioxide then attaches to the resulting tyrosyl radical. What remains is 3-nitrotyrosine, a lasting mark on the protein. It can alter the protein's work. Source 4, 3↓ depletes The nitro group changes the charge and shape of tyrosine. Depending on the protein, this can inhibit or redirect its work and stop a phosphate group from being attached at this site. observed in studies Source 3, 4
   ⚖ When the balance tips

   too much — If 3-nitrotyrosine accumulates in proteins, more of the affected proteins work in an altered way, and more marked proteins go into breakdown.

   too little — If little 3-nitrotyrosine forms, the tyrosine residues remain free for their usual role, for example as sites for phosphate groups.

   observed in studies · Source 3, 4

5. Free 3-nitrotyrosine → NHPA Via intermediate steps, this gives rise to 3-nitro-4-hydroxyphenylacetic acid. It is the main breakdown product of free nitrotyrosine. In this form most of it leaves the body. Source 6↓ depletes The conversion to NHPA turns free nitrotyrosine into a form that the kidney excretes. In this way most of it leaves the body not as nitrotyrosine. observed in studies Source 6
   ⚖ When the balance tips

   too much — If a lot of NHPA forms, it does not come from nitrotyrosine alone: in studies it also arises from nitration of the body's own para-hydroxyphenylacetic acid.

   too little — If little nitrotyrosine is converted to NHPA, a larger share leaves the body unchanged as nitrotyrosine.

   observed in studies · Source 6

6. NHPA → Excretion in urine The kidney releases NHPA and a smaller share of unchanged nitrotyrosine into the urine. With this the breakdown products leave the body. Source 6↓ depletes NHPA and nitrotyrosine leave the body with the urine. This ends the path of nitration; the substances enter no further reaction. observed in studies Source 6
   ⚖ When the balance tips

   too much — If more nitrotyrosine is released, the kidney passes more NHPA and unchanged nitrotyrosine into the urine.

   too little — If little nitrotyrosine is released, the kidney passes correspondingly little NHPA and nitrotyrosine into the urine.

   observed in studies · Source 6

Further stations

• L-arginine — amino acid L-arginine is the starting material for nitric oxide. It supplies the nitrogen atom that later sits in the molecule. NO synthase converts it into NO and citrulline. Source 1↑ supplies Arginine is the only starting material of the NO synthases: they oxidise one of its nitrogen atoms to NO, leaving citrulline. Without arginine no NO forms by this route. established physiology Source 1
  ⚖ When the balance tips

  too much — If a lot of arginine is present, NO synthase is already largely supplied with its starting material; how much NO forms is then decided mainly by calcium and BH4.

  too little — If little arginine is in the cell, NO synthase cannot fully convert the oxygen; in studies it then releases more superoxide, as it does when BH4 is scarce.

  observed in studies · Source 1, 2

• Nitrite — intercepted peroxynitrite Peroxiredoxins and glutathione peroxidases remove an oxygen atom from peroxynitrite. This forms nitrite, which is further converted to nitrate and excreted via the kidney. In this way less peroxynitrite reaches proteins. Source 5↓ depletes Peroxiredoxins and glutathione peroxidases capture peroxynitrite before it nitrates tyrosine. To do so they use up electrons from thioredoxin and glutathione. observed in studies Source 5
  ⚖ When the balance tips

  too much — If the peroxidases capture a lot of peroxynitrite, more thioredoxin and glutathione are oxidised, which the cell must reduce again with NADPH.

  too little — If little selenium or glutathione is available, the glutathione peroxidases capture less peroxynitrite, and more of it reaches the tyrosine residues of proteins.

  observed in studies · Source 5

• Free 3-nitrotyrosine — after protein breakdown When marked proteins are broken down in the proteasome and in lysosomes, 3-nitrotyrosine is released as a single amino acid. It is not built back into proteins. In this way the mark leaves the protein stock. Source 6, 3↓ depletes By breaking down marked proteins, the cell removes the nitration from its protein stock. The released 3-nitrotyrosine is not reused but broken down further. observed in studies Source 6, 3
  ⚖ When the balance tips

  too much — If many marked proteins are broken down, more free 3-nitrotyrosine is released, and more NHPA forms.

  too little — If marked proteins are broken down slowly, 3-nitrotyrosine stays bound in the proteins longer, and less of it is released.

  observed in studies · Source 6, 3

Cofactors in this pathway

• Arginine — Starting material of the NO synthases; the nitrogen atom of NO comes from it; arginine becomes citrulline in the process Source 1In the ORY catalogue as a laboratory value: Arginin
• Tetrahydrobiopterin (BH4) — Cofactor of the NO synthases; without it they release superoxide instead of NO; superoxide and NO form peroxynitrite Source 2
• Iron — Central atom of the haem in the oxygen-binding part of the NO synthases; there it binds and activates oxygen Source 1In the ORY catalogue as a laboratory value: Eisen
• NADPH — Electron source of the NO synthases and of the NADPH oxidases; the oxidases use it to make superoxide Source 1, 7
• FAD and FMN — Pass electrons from NADPH to the haem in the reductase part of NO synthase for NO formation Source 1
• Calcium — Binds to calmodulin; only this complex sets the NO synthases going; so NO forms when needed Source 1In the ORY catalogue as a laboratory value: Calcium (intrazellulär)
• Selenium — Building block of the glutathione peroxidases that remove an oxygen atom from peroxynitrite; this yields nitrite Source 5In the ORY catalogue as a laboratory value: Selen
• Glutathione — Provides the glutathione peroxidases with the electrons for this step; it is itself oxidised in the process Source 5In the ORY catalogue as a laboratory value: Glutathion (GSH)