Detection of Elevated Level of Tetrahydrobiopterin in Serum Samples of ME/CFS Patients with Orthostatic Intolerance: A Pilot Study 2023,Gottschalk ea

Sly Saint

Senior Member (Voting Rights)
Abstract
Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a multisystem chronic illness characterized by severe muscle fatigue, pain, dizziness, and brain fog. Many patients with ME/CFS experience orthostatic intolerance (OI), which is characterized by frequent dizziness, light-headedness, and feeling faint while maintaining an upright posture. Despite intense investigation, the molecular mechanism of this debilitating condition is still unknown. OI is often manifested by cardiovascular alterations, such as reduced cerebral blood flow, reduced blood pressure, and diminished heart rate.

The bioavailability of tetrahydrobiopterin (BH4), an essential cofactor of endothelial nitric oxide synthase (eNOS) enzyme, is tightly coupled with cardiovascular health and circulation. To explore the role of BH4 in ME/CFS, serum samples of CFS patients (n = 32), CFS patients with OI only (n = 10; CFS + OI), and CFS patients with both OI and small fiber polyneuropathy (n = 12; CFS + OI + SFN) were subjected to BH4 ELISA. Interestingly, our results revealed that the BH4 expression is significantly high in CFS, CFS + OI, and CFS + OI + SFN patients compared to age-/gender-matched controls. Finally, a ROS production assay in cultured microglial cells followed by Pearson correlation statistics indicated that the elevated BH4 in serum samples of CFS + OI patients might be associated with the oxidative stress response. These findings suggest that the regulation of BH4 metabolism could be a promising target for understanding the molecular mechanism of CFS and CFS with OI.

IJMS | Free Full-Text | Detection of Elevated Level of Tetrahydrobiopterin in Serum Samples of ME/CFS Patients with Orthostatic Intolerance: A Pilot Study (mdpi.com)
 
https://www.omfcanada.ngo/genetic-and-metabolic-markers-of-bh4-deficiency-in-long-covid/

We have determined that individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome are more likely to have pathogenic variants in BH4 synthesis genes compared to the general population, and we have also identified a corroborating metabolic signature that predicts BH4 deficiency. Oxidative stress during infection can also lead to BH4 deficiency, so there are many potential routes to the same problem. We propose to test whether these genetic markers and other indicators of BH4 deficiency are also present in subjects with Long Covid. If we are successful, we will show that ME/CFS and Long Covid have a common underlying disease mechanism that includes deficiency of BH4.
 
From Wiki:
Tetrahydrobiopterin deficiency (THBD, BH4D) is a rare metabolic disorder that increases the blood levels of phenylalanine. Phenylalanine is an amino acid obtained normally through the diet, but can be harmful if excess levels build up, causing intellectual disability and other serious health problems. In healthy individuals, it is metabolised (hydroxylated) into tyrosine, another amino acid, by phenylalanine hydroxylase. However, this enzyme requires tetrahydrobiopterin as a cofactor and thus its deficiency slows phenylalanine metabolism.

My blood levels of phenylalanine and tyrosine were the lowest markers on my tests.
 
Tetrahydrobiopterin (BH4) is an endogenous cofactor for some enzymatic conversions
of essential biomolecules, including nitric oxide, and monoamine neurotransmitters, and for the
metabolism of phenylalanine and lipid esters.

Over the last decade, BH4 metabolism has emerged as a promising metabolic target for negatively modulating toxic pathways that may result in cell death.

Strong preclinical evidence has shown that BH4 metabolism has multiple biological roles beyond its
traditional cofactor activity. We have shown that BH4 supports essential pathways, e.g., to generate
energy, to enhance the antioxidant resistance of cells against stressful conditions, and to protect from
sustained inflammation, among others.

Therefore, BH4 should not be understood solely as an enzyme cofactor, but should instead be depicted as a cytoprotective pathway that is finely regulated by the interaction of three different metabolic pathways, thus assuring specific intracellular concentrations.

Here, we bring state-of-the-art information about the dependency of mitochondrial activity upon the
availability of BH4, as well as the cytoprotective pathways that are enhanced after BH4 exposure.
We also bring evidence about the potential use of BH4 as a new pharmacological option for diseases
in which mitochondrial disfunction has been implicated, including chronic metabolic disorders,
neurodegenerative diseases, and primary mitochondriopathies.


Pub Date: 2023-05-03
https://www.researchgate.net/public...rin_Beyond_Its_Traditional_Role_as_a_Cofactor
 
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If BH4 expression is high it seems it might be protective of something. They hint at this with oxidative stress, but it might not be that, or not only that. If expression is high but we can confirm deficiency then it might be that its not being expressed enough or that its being over-utilized. Too many maybes, we need more data, though I have not been keeping up to date on any of this.
 
According to the paper tetrahydrobiopterin (BH4) has not been tested in CFS yet. It is involved in amino acid metabolism and the production of nitric oxide (NO) in endothelium.

These seem to be the main results:

upload_2023-6-3_11-2-2.png
 
https://twitter.com/user/status/1695218335535824929




Regulation of BH4 is a very conserved process in a healthy cell. Deficiency of BH4 is mostly genetic and partly metabolic. BH4 deficiency can be diagnosed with increased phenylalanine, phenylketonuria, dopamine loss, and hypocitrulinemia. None of these defects was found in ME.

Looks like these tweets may have been prompted by @LarsSG
 
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The ME/CFS patient population was enrolled based on the Canadian 2003 and Fakuda criteria for ME/CFS. They also met the diagnostic criteria for SCID (ME/CFS’s new name established by the Institute of Medicine). Controls were age/gender matched to CFS subjects who were also invited along with patients in Dr. Peterson’s Internal Medicine Clinic.
A couple of typos, some odd phrasing and missing references. This version looks a bit like a preprint.

There's quite a lot of overlap in the BH4 levels, although a substantial subgroup of the people with ME/CFS do seem to have higher levels of BH4 in serum.

The assay was double blinded.
Interesting about the possible impacts on mitochondrial function:
First, BH4 upregulation is reported to cause mitochondrial impairment of energy metabolism via inhibition of complex I and IV of the electron transport chain, which results in the reduction in mitochondrial membrane potential, the release of cytochrome C and apoptosis [24].



I'm wondering if some sort of drug might be affecting the serum levels - maybe an antidepressant? I don't think the paper reports on the drug intakes of the participants.
There's a 1989 study - Plasma levels of tetrahydrobiopterin and folate in major depression
Plasma levels of tetrahydrobiopterin (BH4) and the related pterin folate were concurrently measured in 20 pairs of depressed patients and age-matched controls. The mean values of plasma BH4 in depressed patients was significantly elevated to a level about 150% of that found in the controls. Folate levels were not different between groups. These findings emphasize that BH4, a required cofactor in the biosynthesis of catecholamines and indolamines, is altered in depression.
So, the 1989 study found that levels of plasma BH4 were increased in depressed patients. Of course we are dealing with all sorts of uncertainties - were the patients actually depressed, or did they just tick the boxes for physical symptoms on the survey?
But, in a 2018 review,
Low BH4 levels have also been found in postmortem brains of subjects with a history of severe depression (Blair et al., 1984).
There seems to be more studies suggesting low blood BH4 is associated with inflammation and depressive symptoms. So, I'm not sure what is going on.

From a 2021 study Tetrahydrobioterin (BH4) Pathway: From Metabolism to Neuropsychiatry the suggestion that BH4 can cause neuropathic pain, but low levels can cause neuron damage. It can be inflammatory and anti-inflammatory. It is suggested that the effect of BH4 depends on the oxidative status of the cell.
Once synthesized, BH4 can act as an anti-inflammatory molecule and scavenger of free radicals protecting against oxidative stress [77-79]. In addition, BH4 is prone to autoxidation in the presence of molecular oxygen, leading to the release of superoxide radicals and therefore contributing to inflammatory processes [80]. Data obtained in murine models of inflammatory and neuropathic pain showed that pharmacological decrease in BH4 synthesis induces analgesia via a reduction of neuronal hyperexcitability and limitation of immune cell proliferation and inflammation, supporting a pro-inflammatory role of BH4 [81, 82]. Lowering BH4 levels using 2,4-Diamino-6-hydroxypyrimidine (DAHP) as a GCH1 inhibitor reduced neuropathic pain in rats following nerve injury and inflammation, confirming the pronociceptive action of excess BH4 production in the somatosensory system [83]. Concomitantly, the sensory responses to non-inflammatory pain (mechanical or thermal) of hph-1 mice deficient in BH4 are unchanged [84]. These results are in agreement with clinical data reporting significant reductions in both pain and inflammation in rheumatoid arthritis and inflammatory bowel disease patients treated with sulfasalazine [81], an inhibitor of SR [85] leading to reduced BH4 levels.

However, other studies have shown that supplementation with BH4 in murine atherosclerotic models reduces vascular inflammation [86-88], leukocyte infiltration, activation of macrophages, and expression of pro-inflammatory cytokines. A downregulation of pro-inflammatory cytokines has been described after BH4 treatment in a murine model of pancreas transplantation [89]. This was associated with a gain in the cell viability of the transplant and prolonged recipient survival. Primary cultures of neurons depleted in BH4 showed increased vulnerability to hypoxia and oxidative damage that lead to exacerbated cell death, illustrating here the antioxidant action of BH4 [90]. Levels of neopterin have been shown to increase after traumatic brain injury or brain infections, indicating a local production by nervous cells, where it is supposed to exert cytoprotective and anti-inflammatory properties [27]. These data rather support here a role for BH4 as an anti-inflammatory molecule.

Overall, the response of BH4 as a pro- or anti-inflammatory molecule is complex and largely depends on the oxidative status of the cell.
 
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Some issues with the controls in this study:
The methods section says that the four cancer patients in the controls were disease positive controls:
The four cancer patients were blindly included in this analysis and served as a disease positive control.
The results section says that the results from the cancer patients were excluded from the analysis.
Four subjects from the control group had cancer-related complications and therefore were excluded from the final analysis.

Table 1 lists 34 controls, 4 of whom have cancer.

So, 34 controls, but only 30 are used, which is ok, although it would be good to see the levels of those 4 with cancer.

The text reports a statistical analysis with 28 controls.
Interestingly, a double-blinded ELISA analysis followed by a non-parametric Mann–Whitney Utest [* p < 0.05 (=0.033); U = 304.5] revealed that the levels of BH4 were significantly higher in n = 32 CFS patients compared to n = 28 control subjects (Figure 1A).

The caption for Figure 1 talks about 30 controls:
(A) Serum samples were 1:2 diluted by assay diluents and then assayed for BH4 by competitive ELISA method. n = 30 control and n = 32 CFS subjects were included. The significance of mean was tested by a Mann–Whitney U test between two groups at * p < 0.05.
 
BH4 has a half life of 4 hours in healthy adults. In PBS it has a half life of 16 minutes at room temperature and completely destroyed at 90 minutes.

Guess what Simmaron did to some of their samples. Yep, they used PBS.

Briefly, serum samples were two-times diluted with assay diluent (or 1× PBS) and then pipetted on a microplate that was pre-coated with polyclonal anti-BH4 antibody. After 30 min of incubation with the sample, BH4-HRP conjugate was added to the plate and incubated for an additional 1 h at 37 °C. The residual serum samples were decanted and washed on the plate with 1× wash buffer (alternatively, with 1× PBS-T) three times for five minutes each using a multi-channel pipette.
 
There seems to be more studies suggesting low blood BH4 is associated with inflammation and depressive symptoms. So, I'm not sure what is going on.
I think BH4 oxidises with air very quickly so likely papers are useless unless they accounted for that. This 2022 study discusses some of the things the authors needed to implement to stabilise BH4 in order to measure it.
Measurement of Tetrahydrobiopterin in Animal Tissue Samples by HPLC with Electrochemical Detection—Protocol Optimization and Pitfalls

BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2
 
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