What’s going on in orthostatic intolerance (symptoms triggered by standing or sitting up) has been well described – from the outside. We can see heart rates go up (postural orthostatic tachycardia syndrome (POTS)), blood pressures drop (neurally mediated hypotension (NMH)) or norepinephrine levels rise (hyperadrenergic POTS), or CO2 levels drop (postural orthostatic syndrome of hypocapnia (POSH).

What’s happening on the inside – at the molecular level – is more of a mystery. Yes, we know about norepinephrine levels, but what is causing them? Studies suggest that a messed-up renin-angiotensin-aldosterone (RAA) pathway contributes to the low blood volume found in ME/CFS and POTS but no one, to my understanding, has figured out, molecularly, how that has happened.

In”Dysregulation of tetrahydrobiopterin metabolism in myalgic encephalomyelitis/chronic fatigue syndrome by pentose phosphate pathway“, the Simmaron Research Foundation’s small but creative research team led by Avik Roy and Gunnar Gottschalk is trying to get at the molecular roots at them – and in ME/CFS to boot.

The Study

It revolves around a compound called tetrahydrobiopterin (BH4) that plays a crucial role in several major processes. At the right levels, BH4 allows for the production of the feel-good brain chemicals dopamine and serotonin, assists with mitochondrial functioning, and helps the blood vessels to vasodilate or open.

High levels of BH4 have been associated with all sorts of negative results including mitochondrial dysfunction, cognitive problems, immune dysregulation, inflammatory and autoimmune diseases, and chronic pain.

When we last heard from the Simmaron research team, they’d found high BH4 levels in ME/CFS patients with orthostatic intolerance – an outcome that had never been found before.

Another Balky Metabolic Pathway?

Enter the pentose phosphate pathway (PPP). The PPP is a metabolic pathway that runs parallel to glycolysis and is activated during high levels of oxidative stress – which we know is present in ME/CFS. The pathway produces something called NADPH to regenerate the key antioxidant in our body – glutathione – when glutathione levels dip low. Several studies have made it clear that glutathione levels in the brain are low in ME/CFS.

The pathway can also kick in when glycolysis is not functioning. The status of glycolytic functioning in ME/CFS is unclear, as some studies suggest it’s functioning just fine while others suggest it is not.

The PPP contains two phases: an oxidative (antioxidant enhancing) phase, and a non-oxidative phase that synthesizes sugars and produces something called ribose-5-phosphate – which plays a critical role in the production of BH4.

Finding high BH4 levels in ME/CFS, the Simmaron researchers dug into the pentose phosphate pathway to see what was going on. Multiple tests (gene expression study, real-time PCR, and quantitative ELISA analyses) indicated that the non-oxidative side of the PPP pathway – the side that produces BH4 – had been highly upregulated in ME/CFS.

Low Cellular Oxygen Levels Tagged

And what an interesting upregulation it was. The increased expression of genes involved in lactate production and the non-oxidative side of the pathway suggested that low oxygen levels in the cells of ME/CFS patients were triggering the non-oxidative side of the PPP to gear up.

As that was happening, the downregulated oxidative side of the PPP likely resulted in increased oxidative stress (check!), impaired lipid synthesis (check!), tissue repair (after exercise???), and pathogen killing (check!).

The possibility that low oxygen levels, or hypoxia, figure in ME/CFS has been around for quite a while. They tested the hypothesis that low oxygen levels were causing the pentose phosphate pathway to produce increased BH4 levels by creating a hypoxic state and exposing cells to it. The results suggested it was.

Turning a Good Enzyme Bad

In the presence of oxidative stress – which we know is plentiful in ME/CFS – BH4 does something particularly nasty: it turns a “good” enzyme – eNOS – into a “bad” enzyme (iNOS). In its “good” form, eNOS helps to open the blood vessels and keep them healthy.

In its “bad” form, iNOS is associated with the production of reactive nitrogen species (RNS) such as peroxynitrite, activation of the microglial cells in the central nervous system (neuroinflammation), excitotoxicity in the central nervous system, and mitochondrial dysfunction. (Ouch!)

Neuroinflammation Too?

Indeed, when the Simmaron team investigated the impact of BH4 upregulation on the microglial cells and inflammation, they found that under hypoxic conditions, BH4 triggered the production of nitrite – a reactive nitrogen species (a free radical) but only in the ME/CFS patients’ cells. That suggested the elevated BH4 levels in ME/CFS patients’ cells may be triggering neuroinflammation.

Bad Breakdown Product

Further analyses indicated that two byproducts of BH4 breakdown – BH2 and DHPR – were strongly upregulated in the ME/CFS patients as well – and here we come back to the possible molecular origins of orthostatic intolerance.

It turns out that high BH2 levels can produce reduced levels of eNOS – the form of the enzyme that opens the blood vessels. High BH2 levels could also increase platelet activation, cause damage to the endothelial cells lining the blood vessels, and increase the risk of atherosclerosis and blood clotting.

Nasty Oxidative Stress Mix

In fact, it’s worse than that. In the presence of high BH2 levels, instead of producing endothelial nitric oxide, eNOS produces a free radical called superoxide. Instead of opening the blood vessels as it normally does, eNOS does a Jekyll/Hyde about-face and actually ends up clamping down on them.

When superoxide pairs with nitric oxide, it produces peroxynitrite – a dangerous free radical that loves to poke holes in cellular membranes, cause mitochondrial dysfunction, damage our DNA, etc. Peroxynitrite is difficult to measure in the blood, but ample evidence of oxidative stress has been found in ME/CFS. Martin Pall’s 2001 ME/CFS hypothesis proposed that chronically high levels of peroxynitrite were driving the disease, and Bindu Paul and Marian Lemle more recently suggested that oxidative stress plays a key role in it and long COVID.

The high BH2 levels the Simmaron group found in ME/CFS, then, are potentially a big deal.

Potential Therapeutic Interventions

The authors did not mention treatment possibilities, but AI ChatGpt had some ideas about treating the variety of potential problems this study found.

Improve eNOS activity

• Eat more dietary nitrates found in beetroot and green leafy vegetables
• Supplement with omega-3 fatty acids, L-Arginine (eNOS substrate – but rapidly metabolized), L-Citrulline (precursor to L-arginine – possibly more effective that L-arginine), antioxidants (Vit. C, E and NAC), nicotinamide riboside, CoQ10, tetrahydrobiopterin (BH₄) (questionable given high BH4 findings?)
• Try statins, metformin, phosphodiesterase-5 (PDE5) Inhibitors (e.g., sildenafil).

Reduce iNOS activity

• Try Resveratrol, quercetin, and curcumin
• Anti-inflammatories (NSAIDs) and TNF-a inhibitors (etanercept), (anti-TNF therapy) or anakinra (IL-1 receptor antagonist)
• Experimental drugs – L-NIL (N6-(1-Iminoethyl)-L-lysine) and aminoguanidine.

Reduce BH2 levels

• Antioxidants to combat oxidative stress can prevent the oxidation of BH4 to BH2, maintaining the proper BH4/BH2 balance
• Antiplatelet therapy like aspirin can counteract the pro-clotting environment
• Supplement with folate to enhance BH4 regeneration from BH2
• Supplement with N-acetylcysteine (NAC) to increase glutathione levels.

Conclusion

One of the nice things about this study – done at the cellular level – is how well its findings fit with what we know about ME/CFS. High levels of oxidative stress, low cellular oxygen levels, low glutathione levels,  inflammation, neuroinflammation, and problems with blood vessel flows and lipids – these all seem to be part and parcel of ME/CFS.

A dysregulated pentose phosphate pathway, and the BH4, and BH2 connection provide another explanation for them. Will it turn out? The studies are small and only time will tell if they’re on the right track.

The authors have proposed a way to find out, though. A high-resolution metabolomics study using a large sample size that was informed by machine learning-based network analyses they believe would confirm or deny the role that PPP-induced BH4 plays in ME/CFS.

The small Simmaron research team with their ME/CFS mouse model work, autophagy, rapamycin clinical trial, and now the BH4, pentose pathway, and BH2 findings has been uncommonly productive. They appear to have the samples they need to do the metabolomic project – all they need at this point is the funding. May they get it!

• Coming up – an update on the Simmaron Research Foundation’s ME/CFS Rapamycin clinical trial

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This study was a toughie! It’s biochemistry-oriented and took a lot of work to understand – particularly for someone who barely made it through organic chemistry in college (lol) – and for readers to get through. It’s at the molecular level, though, that understanding this disease will probably bring us the greatest breakthroughs. I’m committed to understanding (as best I can) this disease and communicating it.

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