2-OGDO enzymer og jern redox status hos ME patienter

I de foregående tre blogindlæg har jeg redegjort for, at cellers jern redox status er vigtig for aktiviteten af en række enzymer.

Enzymgruppen 2-oxoglutarate dependent dioxygenaser (2-OGDO) anvender også jern i ferro form (Fe2+) som cofactor.

Jeg vil nu stille spørgsmålet: Er 2-OGDO enzym aktiviteterne i ME celler påvirket af en forstyrret jern redox ligevægt?

2-OGDO enzymerne katalyserer en lang række biologiske processer, herunder (1):

• hydroxylering af kollagen
• demethylering af histoner
• første og sidste trin i dannelsen af carnitin
• nedbrydning af fytinsyre (eng: phytanic acid)

Aktivering af 2-OGDO enzymer starter med binding af Fe2+, hvorefter ilt (dioxygen = O2) bindes til Fe2+. Dette stimulerer den oxidative decarboxylering af 2-oxoglutarate (=alfa-ketoglutarate) til succinate og CO2, og samtidig dannes oxiderede jern mellemprodukter (Fe3+/4+). Disse højvalente jern oxidanter kan hydroxylere et molekyle, dvs sætte en OH-gruppe på molekylet (1).

Ascorbat (c-vitamin) og glutathione kan reducere Fe3+ og Fe4+ tilbage til Fe2+, således at aktiviteten af 2-OGDO enzymerne genetableres. Bemærk at genetablering af ferro-tilstanden (Fe2+) også er det centrale i de tre forrige blogindlæg.

Citat fra reference 1: "It seems that the 2-OGDO enzymes are at the nexus of independent control between energy metabolism and availability of oxygen and iron in the regulation of cellular homeostasis."

Ydermere oplyser ref 1, at transferrin og transferrin receptor er nøgle-komponenter til regulering af jern ligevægten. Disse komponenter dukkede op i et ME genomic studie:

OMICS and iron metabolism in ME
http://followmeindenmark.blogspot.com/2019/10/omics-and-iron-metabolism-in-me.html

Dannelse af carnitin og ME sygdomsmekanismen

Carnitin er nødvendig for omsætning af fedtsyrer. Carnitin fås gennem kosten, og kroppen danner også selv carnitin. Carnitinen dannes gennem flere enzymatiske trin, hvor to trin katalyseres af 2-OGDO enzymerne: trimethyllysine hydroxylase (TMLH) og gamma-butyrobetaine hydroxylase (BBOX eller GBBH), hvor Fe2+ er cofactor (3).

Fedtsyreforbrændingen er påvirket hos ME patienter, og der er fundet lave plasma værdier af carnitin hos patienterne. Læs blogindlæg:

ME minder om medfødt metabolisk defekt
http://followmeindenmark.blogspot.com/2019/02/me-minder-om-medfdt-metabolisk-defekt.html

Plasma niveauet af alfa-ketoglutarate var forhøjet hos ME patienter i Germain et al's metabolisme studie (4). Hvordan hænger det hele sammen?

Referencer

1) Salminen et al: 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process Cell Mol Life Sci. 2015 Oct;72(20):3897-914. doi: 10.1007/s00018-015-1978-z. Epub 2015 Jun 29. https://www.ncbi.nlm.nih.gov/pubmed/26118662

2) Loenarz and Schofield: Expanding chemical biology of 2-oxoglutarate oxygenases. Nat Chem Biol, 2008, 4, 3. https://www.ncbi.nlm.nih.gov/pubmed/18277970

3) Carnitine biosynthesis https://en.wikipedia.org/wiki/Carnitine_biosynthesis

4) Germain et al: Prospective Biomarkers from Plasma Metabolomics of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Implicate Redox Imbalance in Disease Symptomatology. Metabolites. 2018 Dec 6;8(4). pii: E90. doi: 10.3390/metabo8040090.https://www.ncbi.nlm.nih.gov/pubmed/30563204

REDOX og NO-sGC-cGMP-stivejen

Der findes en gammel hypotese om, at NO-sGC-cGMP-stivejen og hermed blodgennemstrømningen er påvirket hos ME patienter (1).

Nitrogenoxid (NO) er et gasformigt signalmolekyle, der har flere forskellige funktioner i kroppen. Soluble guanylate (eller guanylyl) cyclase (sGC) er receptor for NO. At den er soluble (opløselig) betyder, at den er intracellulær. På sGC molekylet findes et hæm-bindende område, der kan binde det gasformige molekyle NO. Hermed kan sGC producere guanosine 3’,5’-monophosphate (cGMP) fra guanosine triphosphate (GTP). Dette kaldes for NO-sGC-cGMP stivejen, som er relevant for kontrol af en lang række fysiologiske processer. F.eks. vil stimulation af sGC få glat muskulatur omkring blodkar til at slappe af. Der er udviklet lægemidler, der kan modulere sGC. De er målrettet tilstande, hvor dannelse af NO og NO tilgængelighed er mangelfuld (1).

På samme måde som hæmoglobin og IDO kan binde ilt via Fe2+ (læs to forrige blogindlæg), så kan sGC i ferro form sGC(Fe2+) binde NO. Når sGC bliver oxideret til sGC(Fe3+) kan NO ikke bindes.

• Aktiv ferro soluble Guanylate Cyclase: sGC(Fe2+) kan binde NO
• Inaktive ferri soluble Guanylate Cyclase: sGC sGC(Fe3+), kan IKKE binde NO

Celler i vaskulær glat muskulatur kan reducere den inaktive sGC(Fe3+) via cytochrome b5 reductase (CYB5B3), også kendt som methæmoglobin reductase. Hermed gendannes den NO-bindende sGC(Fe2+), se figur i reference 2 via link:

Schematic of Cyb5R3 regulating sGC heme redox state
Illustration shows Cyb5R3 sensitizes sGC to NO by reducing the sGC heme iron, controlling cGMP production and vessel relaxation.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527687/figure/F7/

Læs også:

Forhøjet methæmoglobin og "rod i redox" hos ME patienter
http://followmeindenmark.blogspot.com/2019/10/forhjet-methmoglobin-og-rod-i-redox-hos.html

REDOX status påvirker IDO enzym aktivitet
http://followmeindenmark.blogspot.com/2019/10/redox-status-pavirker-ido-enzym.html

Ergenor et al. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential. Nature Reviews. Drug Discovery. Vol 5, sept. 2006
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225477/

Referencer

1) Blogindlæg af 24. marts 2016. Dysregulering af NO-sGC-cGMP-stivejen i ME/CFS http://followmeindenmark.blogspot.com/2016/03/dysregulering-af-no-sgc-cgmp-stivejen-i.html

2) Rahaman et al: Cytochrome b5 Reductase 3 Modulates Soluble Guanylate Cyclase Redox State and cGMP Signaling
Circ Res. 2017 Jul 7;121(2):137-148. doi: 10.1161/CIRCRESAHA.117.310705. Epub 2017 Jun 5.
https://www.ncbi.nlm.nih.gov/pubmed/28584062

REDOX status påvirker IDO enzym aktivitet

I forrige blogindlæg Forhøjet methæmoglobin og "rod i redox" hos ME patienter lærte vi, at hæmoglobin skal være i ferro (Fe2+) tilstand for at kunne binde ilt.

Enzymet indolamine-2,3-dioxygenase (IDO) binder ligeledes ilt via jern i ferro (Fe2+) form. IDO omsætter aminosyren tryptofan til N-formylkynurenine. IDO sætter to iltatomer (to ilt = di oxygen) ind i indolringen på plads nr. 2 og 3 i tryptofan (1):

Link til figur af den kemiske reaktion (1):
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280726/figure/sch1/

Ligesom hæmoglobin kan auto-oxideres til methæmoglobin i ferri (Fe3+) form, kan IDO auto-oxidere til en inaktiv ferri (Fe3+) form (2):

• Aktiv ferro indolamine-2,3-dioxygenase:: IDO(Fe2+), kan binde ilt
• Inaktiv ferri indolamine-2,3-dioxygenase: IDO(Fe3+)

I forrige blogindlæg lærte vi, at cytochrome b5 kan give en elektron til methæmoglobin Hgb(Fe3+), så den aktive iltbindende hæmoglobin Hgb(Fe2+) gendannes.

Cytochrome b5 kan også anvendes til at give en elektron til IDO(Fe3+), således at den aktive IDO(Fe2+) gendannes (3).


Et hold forskere har vist, at ud over cytochrome b5 kan polysulfider eller thiol-molekylet 3-mercaptoindol også anvendes til at regenere IDO enzymet, citat fra reference 2:

"To bind molecular oxygen and activate tryptophan, IDO1 must be in the reduced ferrous state, where it returns in its catalytic cycle without the need for additional reducing equivalents. And yet, if no reductant is supplied during multiple turnovers, IDO1 undergoes abortive autoxidation with loss of superoxide to produce the inactive ferric enzyme. The ferric IDO1 produced in this manner persists even in the reducing environment of cells, suggesting that self-limiting autoxidation is an adaption to prevent excessive activity. Further highlighting the importance of redox regulation in tryptophan metabolism, inactive ferric IDO1 is also prone to long-term inactivation through heme-loss, which likely accounts for the majority of cellular IDO1 found in the apo-form. Regeneration and maintenance of active, ferrous IDO1 in cells has been attributed to cytochrome b5. We sought to explore and understand what other cellular reductants might affect the IDO1 redox switch and, by implication, the immune response. Our initial efforts were motivated by recent work describing the hydrogen sulfide-mediated reduction of myoglobin, hemoglobin, heme oxygenase, and porphyrin complexes. Hydrogen sulfide binds to the ferric heme of these proteins to form a hydrosulfido complex that can then lead to reduction to the ferrous heme and production of sulfhydryl radical, a paradigm matching the requirements for IDO1 activation in cells. The final oxidized products of these reactions, polysulfides, have also gained attention with the discovery of their high, micromolar concentrations within cells and their remarkably versatile reactivity, including ferric heme reduction in cytochrome c."


Hvis hypotesen om ME-IDO-metabolic-trap er sand, kan polysulfider eller 3-mercaptoindol så anvendes til behandling af ME?


Har ME patienter et generelt redox reduktions-potentiale problem?

Læs også:

ME hypotese: The Metabolic Trap - den metaboliske fælde

The IDO Metabolic Trap Hypothesis for the Etiology of ME/CFS

Referencer

1) Efimov et el. The mechanism of substrate inhibition in human indoleamine 2,3 dioxygenase. J Am Chem Soc. 2012 Feb 15;134(6):3034-41. doi: 10.1021/ja208694g. Epub 2012 Feb 2.https://www.ncbi.nlm.nih.gov/pubmed/22299628

2) Nelp et el: Potent activation of indoleamine 2,3-dioxygenase by polysulfides. J Am Chem Soc. 2019 Sep 25;141(38):15288-15300. doi: 10.1021/jacs.9b07338. Epub 2019 Sep 11. https://www.ncbi.nlm.nih.gov/pubmed/31436417

https://pubs.acs.org/doi/10.1021/jacs.9b07338

https://pubs.acs.org/doi/suppl/10.1021/jacs.9b07338/suppl_file/ja9b07338_si_001.pdf

3) Maghzai et al: Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2,3-dioxygenase in human cells. J Biol Chem. 2008 May 2;283(18):12014-25. doi: 10.1074/jbc.M710266200. Epub 2008 Feb 25. https://www.ncbi.nlm.nih.gov/pubmed/18299324

Forhøjet methæmoglobin og "rod i redox" hos ME patienter

Konklusionen på Germain et al (2018) metabolisme studiet var, at det er "rod i redox" (1):
"Our hypothesis is that a disturbance in the redox status influences the status of chemical reaction donors and acceptors as well as their coenzymes such as NAD+/NADH, FAD+/FADH for dehydrogenases. Oxidases would obviously also be affected as catalyzers of redox reactions."

Ordet redox er en sammentrækning af reduktion og oxidation. Det handler om at flytte elektroner mellem kemiske forbindelser.

NAD+/NADH er et eksempel på et redox-par:

• NAD+ kan modtage en elektron
• NADH kan donere en elektron

Jern (latin: Ferrum) bliver også anvendt til kroppens redox-processer:

• Fe3+ er den trivalente jern-ion, også kaldet jern i ferri form (engelsk: ferric state). Fe3+ kan modtage en elektron.
• Fe2+ er den divalente jern-ion, også kaldet jern i ferro form (engelsk: ferrous state). Fe2+ kan donere en elektron.

Jern redox enzymer katalyserer skiftet mellem ferri og ferro formen:

• Ferrireduktaser reducerer Fe3+ til Fe2+
• Ferrooxidaser oxiderer Fe2+ til Fe3+

Hæmoglobin transporterer ilt (også kaldet oxygen eller O2) rundt i kroppen. Hæmoglobin indeholder jern. For at hæmoglobin kan binde ilt, skal jern være i ferro form (Fe2+). Hæmoglobin auto-oxiderer meget langsomt (ca. 3% pr. dag) til methæmoglobin, som er hæmoglobin i ferri form (Fe3+). Methæmoglobin kan ikke binde ilt (2):

• Hæmoglobin: Hgb(Fe2+), kan binde ilt
• Methæmoglobin: Hgb(Fe3+), kan IKKE binde ilt

Hvis niveauet af methæmoglobin bliver for højt, bliver man træt og motionsintolerant, fordi der ikke bliver båret ilt nok rundt i kroppen.

Som kompensation for dannelse af methæmoglobin har kroppen et enzym cytochrome b5, som reducerer methæmoglobin til hæmoglobin. Dvs. cytochrome b5 giver en elektron til methæmoglobin (Fe3+), så der dannes hæmoglobin (Fe2+), som igen kan binde ilt.

Idet cytochrome b5 har en elektron at give bort, er den i ferro form, og kaldes også for ferrocytochrome b5. Når ferrocytochrome br har givet sin elektron bort, bliver den til ferricytochrome b5.

Ferricytochrome b5 skal nu "genoplades" med en ny elektron for at blive til ferrocytochrome  b5. Cytochrome b5 får sin elektron fra NADH (3):

NADH + H⁺ + 2 ferricytochrome b₅ → NAD⁺ + 2 ferrocytochrome b₅


Enzymet der reducerer ferricytochrome b5 til ferrocytochrome b5 hedder cytochrome b5 reduktase, også kaldet methæmoglobin reduktase.

Germain et al (2018) henviser til et ældre studie, hvor der er målt forhøjede niveauer af methæmoglobn hos ME patienter (1):
"Richards et al.found that methaemoglobin was one of the principal components that differentiated their ME/CFS patients and control cohorts. This hemoglobin carries the oxidized form of the iron ion, namely, the ferric state instead of the ferrous state necessary for the hemoglobin to bind oxygen. Even though methaemoglobin measurements are not part of our dataset, it is intriguing to relate its oxidation state to a disturbed redox environment while the effect of the inability to bind oxygen could translate into anoxia and asphyxia."


I Richards et al's studiet står der (4):

Methaemoglobin was found to be the major component associated with variation in symptom expression in CFS patients (R(2) = 0.99, P <0.00001), which included fatigue, musculoskeletal symptoms, pain and sleep disturbance.

Måling af methæmoglobin

Det er let at måle methæmoglobin. Man tager en blodprøve og sprøjter den direkte ind i blodgas apparatet ABL800 og to minutter senere har man resultatet. Ethvert hospital har et blodgas apparat.

ABL800:
https://www.radiometer.dk/da-dk/videnscenter/h%C3%A5ndb%C3%B8ger-og-apps/acute-care-testing-handbook/critical-parameters-in-acute-care-testing

Referencer

1) Germain et al: Prospective Biomarkers from Plasma Metabolomics of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Implicate Redox Imbalance in Disease Symptomatology. Metabolites. 2018 Dec 6;8(4). pii: E90. doi: 10.3390/metabo8040090.
https://www.ncbi.nlm.nih.gov/pubmed/30563204

2) Umbreit: Methemoglobin--it's not just blue: a concise review. Am J Hematol. 2007 Feb;82(2):134-44. https://www.ncbi.nlm.nih.gov/pubmed/16986127

3) Wikipedia. Cytochrome b5 https://en.wikipedia.org/wiki/Cytochrome_b5

4) Blood parameters indicative of oxidative stress are associated with symptom expression in chronic fatigue syndrome. Richards RS, Roberts TK, McGregor NR, Dunstan RH, Butt HL
Redox Rep. 2000; 5(1):35-41.https://www.ncbi.nlm.nih.gov/pubmed/10905542

En ny biosensor viser, at SS-31 er et potentielt lægemiddel til ME

Dr. Ron Davis fra Standford Universitet og hans team har udviklet en nano-elektrisk biosensor (på engelsk populært kaldet "the nanoneedle"). Den kan vise om immunceller fra en blodprøve kommer fra ME patienter eller fra raske kontrolpersoner.

Immunceller fra både ME patienter og raske kontrolpersoner udsættes for salt-stress (også kaldet osmotisk stress) i biosensoren ved, at der tilsættes en natriumklorid-opløsning. Biosensoren måler et elektrisk signal (impedansen), som er et resultat af celleprocesser udløst af salt-stress.

Forskningen er beskrevet i denne artikel:
A nanoelectronics-blood-based diagnostic biomarker for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)
https://www.pnas.org/content/116/21/10250.long

Dr. Ron Davis forsøger nu at finde et lægemiddel, der kan normalisere ME-cellers respons på salt-stress. Han afprøver forskellige lægemidler ved at tilsætte dem til blod fra ME patienter i biosensoren.

To lægemidler får celler fra ME blodprøver til at opføre sig normalt i biosensoren. Disse resultater blev fremlagt på Havard Symposium 2019:

Dr. Ron Davis Presents on ME/CFS Research Activities at Stanford at Inaugural Harvard Symposium

https://www.youtube.com/watch?v=02TUcGqTFAI&t=37s

Det ene lægemiddel er:  

Copaxone med indholdsstoffet hedder Glatirameracetate. Læs om det her: http://pro.medicin.dk/Medicin/Praeparater/2889

Det andet lægemiddel, som jeg synes er mest interessant, er:

SS-31, Szeto-Schiller Peptide 31, Elamipretide. Lægemidlet er endnu ikke godkendt, men der arbejdes med det på forsøgsbasis. Læs om det her: 

Mitochondrial dysfunction and oxidative stress in metabolic disorders - A step towards mitochondria based therapeutic strategies
https://www.ncbi.nlm.nih.gov/pubmed/27836629

The Mitochondrial Antioxidant SS-31 Modulates Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Type 2 Diabetes
https://www.ncbi.nlm.nih.gov/pubmed/31466264

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice
https://www.ncbi.nlm.nih.gov/pubmed/30597195

The mitochondrial antioxidant SS-31 increases SIRT1 levels and ameliorates inflammation, oxidative stress and leukocyte-endothelium interactions in type 2 diabetes
https://www.ncbi.nlm.nih.gov/pubmed/30367115

SS-31 Provides Neuroprotection by Reversing Mitochondrial Dysfunction after Traumatic Brain Injury
https://www.ncbi.nlm.nih.gov/pubmed/30224944

From the muscle hypothesis to a muscle solution?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437437/

Novel Mitochondria-Targeting Peptide in Heart Failure Treatment: A Randomized, Placebo-Controlled Trial of Elamipretide
https://www.ncbi.nlm.nih.gov/pubmed/29217757

Therapies for mitochondrial diseases and current clinical trials
https://www.ncbi.nlm.nih.gov/pubmed/28943110

OMICS and iron metabolism in ME

Genomics in ME

Gene variants of TF, TFRC, and HPX have been identified as risk loci in ME patients (1):




Slide from: Whole Genome Sequencing and Analysis of ME/CFS
https://www.youtube.com/watch?v=nIJX-Q7w_Z4


Serotransferrin (usually just called transferrin), TF: iron-binding protein

Transferrin receptor, TFRC: plasma membrane protein that allows cellular uptake of iron-loaded transferrin

Hemopexin, HPX: a protein that binds free heme (or met-heme) and transport it to the liver for break-down and iron recovery.

Haptoglobin, HP: a protein that binds free hemoglobin and transport it to the liver for break-down and iron recovery.

Hemopexin and haptoglobin prevent heme toxicity.

Proteomics in ME

Serotransferrin precursor (gene: TF), number of unique peptides identified in the cerebrospinal fluid (table S1 in ref 2):

1) Controls: 105

2) ME patients: 141

3) Post treatment Lyme patients: 105

Transferrin variant fragment  (gene: TF), number of unique peptides identified in the cerebrospinal fluid (table S1 in ref 2):

1) Controls: 104

2) ME patients: 130

3) Post treatment Lyme patients: 99

Transferrin receptor protein 1 (gene: TFRC), number of unique peptides identified in the cerebrospinal fluid (table S1 in ref 2):

1) Controls: 1

2) ME patients: 2

3) Post treatment Lyme patients: 3

Haptoglobin precursor (gene: HP) , number of unique peptides identified in the cerebrospinal fluid (table S1 in ref 2).

1) Controls: 30

2) ME patients: 39

3) Post treatment Lyme patients: 34

Hemopexin precursor (gene: HPX) , number of unique peptides identified in the cerebrospinal fluid (table S1 in ref 2).

1) Controls: 61

2) ME patients: 197

3) Post treatment Lyme patients: 189

Another proteome study on cerebrospinal fluid from ME patients also showed increased levels of hemopexin and haptoglobin (3).

Metabolomics in ME

A metabolomic study on plasma from ME patients showed increased levels of heme (4).

Methemoglobin in ME

A study showed increased levels of methemoglobin in ME patients. Methemoglobin was found to be the major component associated with variation in symptom expression in ME patients (R(2) = 0.99, P <0.00001), which included fatigue, musculoskeletal symptoms, pain and sleep disturbance (5).

Activated macrophages produce an inducible NO synthase (iNOS or NOS2). This NO produced during inflammation and other oxygen reactive species results in conversion of hemoglobin to methemoglobin, and this consequently results in an increased rigidity of the red blood cell (RBC) with increased RBC lysis (6).  

Methemoglobin is an activator of endothelial cells by stimulation of IL-6, IL-8 and E-selectin (6).

Red blood cell deformability is diminished in ME patients (7).

Genomics, proteomics and transcriptomics show the iNOS pathway is upregulated in ME (8).

References:

1) Whole Genome Sequencing and Analysis of ME/CFS https://www.youtube.com/watch?v=nIJX-Q7w_Z4

2) Schutzer et al: Distinct Cerebrospinal Fluid Proteomes Differentiate Post- Treatment Lyme Disease from Chronic Fatigue Syndrome. PLOS One February 2011, volume 6, Issuehttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017287

3) Baraniuk et al: A Chronic Fatigue Syndrome - related proteome in human cerebrospinal fluid. BMC Neurol. 2005 Dec 1;5:22. https://www.ncbi.nlm.nih.gov/pubmed/16321154

4) Germain et al: Prospective Biomarkers from Plasma Metabolomics of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Implicate Redox Imbalance in Disease Symptomatology. Metabolites. 2018 Dec 6;8(4). pii: E90. doi: 10.3390/metabo8040090.
https://www.ncbi.nlm.nih.gov/pubmed/30563204

5) Richards et al: Blood parameters indicative of oxidative stress are associated with symptom expression in chronic fatigue syndrome. Redox Rep. 2000;5(1):35-41. https://www.ncbi.nlm.nih.gov/pubmed/10905542

6) Umbreit: Methemoglobin--it's not just blue: a concise review. Am J Hematol. 2007 Feb;82(2):134-44. https://www.ncbi.nlm.nih.gov/pubmed/16986127

7) Saha et al: Red blood cell deformability is diminished in patients with Chronic Fatigue Syndrome. Clin Hemorheol Microcirc. 2019;71(1):113-116. doi: 10.3233/CH-180469. https://www.ncbi.nlm.nih.gov/pubmed/30594919

8) Genomics, proteomics and transcriptomics show the iNOS pathway is upregulated in ME. https://followmeindenmark.blogspot.com/2019/09/genomics-proteomics-and-transcriptomics.html