Urine Metabolomics Exposes Anomalous Recovery after Maximal Exertion in Female ME/CFS Patients 2023, Glass, Hanson et al

[Hutan]

From Glycine Metabolism and Its Alterations in Obesity and Metabolic Diseases, 2019

Recent evidence suggests that the glycine conjugation pathway is an essential detoxification pathway [61,62,63]. Glycine can be conjugated to various endogenous and xenobiotic metabolites (e.g., benzoate, derivatives of branched chain amino acids (BCAA), β-oxidation intermediates and metabolites of polyphenols), which can be potentially toxic when they accumulate in the organism [62]. The resulting acylglycines are less toxic and more hydrophilic and are excreted in the urine [62,64]. As these metabolites are esterified to CoA, it has been proposed that glycine conjugation contributes to CoA homeostasis, since the reaction releases CoA [64]. Conjugation activity depends on the metabolite and on the enzyme catalyzing the reaction, i.e., acid:CoA ligases and glycine N-acyltransferases (GLYAT) [64,65].

I read that as
A range of metabolites can be toxic if they accumulate. They form esters with CoA, (CoA being a molecule important in the Citric acid cycle i.e. important for producing ATP).
(e.g. for benzoate: ATP + benzoate + CoA

AMP + diphosphate + benzoyl-CoA)
Glycine can react with these esters, releasing the CoA, contributing to CoA homeostasis and simultaneously de-toxifying high levels of the metabolites. The resulting, less-toxic, acylglycines are excreted in the urine.

This excretion seems to be happening in the healthy controls of this study after exercise, and it's quite normal for it to happen in healthy athletes when they do acute exercise too. (So it's not the result of the healthy controls having a higher BMI; this is a normal process. ) In contrast, the women with ME/CFS in this study did not excrete acylglycines in their urine after exertion during the same time period.

I think a delay in or incapacity for acylglycine excretion might mean that it takes longer to clear toxic levels of metabolites, and the CoA that could otherwise be participating in further cranking of the Citric Acid cycle to produce energy is locked up, conjugated to metabolites.

Does that make sense?
(I haven't listened to Dr Katherine Glass's video yet, guess it would be a good idea to do that first. It's just that I was thinking about this when I woke up.)

 

[Sean]

We need an overall authority in charge of ME/CFS research

It is a nice idea, but be careful what you wish for. The BPS club were effectively that authority for decades.

Is it possible that excretion of metabolites could be affected by endothelial dysfunction?

Or vice-versa?

 

[Ravn]

Intriguing study.

Our larger cohort of ME/CFS patients and healthy sedentary controls is BMI-matched, and therefore if this pilot study is expanded, this will not be an issue in the future.

[My bold] So they want to expand their pilot study but don't currently have the funding to do it? Or am I misreading between the lines? Someone, anyone, please give this team a big pot of money!

There's one thing that's tying my brain into knots with studies like this one which point towards a failure of ME cells to mount an appropriate response to exertion. Why don't these studies show any difference at rest/baseline? We're most categorically ill all of the time, albeit more severely with PEM - so surely something must be measurably different already at baseline. Why does this not show up in the metobolomics?

Other studies have led to hypotheses that ME cells are stuck in a cell danger response or that they're exhausted (immune cells) or that they're running at full capacity and with all compensatory mechanisms switched on already at rest, or all of the above, and that the cells therefore are unable to respond to further demand. Which would fit the findings here that ME cells simply do nothing in response to exercise. But I would expect all of the hypotheses to impact metabolism at baseline as well as after exertion. So why do our baseline metabolomes look so seemingly normal? What are we missing? Something the tests aren't measuring? Yet they do throw up all manner of unidentified compounds. Or are the sort of pwME who participate in CPET studies so mild they're actually as well as the general population between episodes of PEM? That sounds unlikely to me.

On a different note, I find the sheer number of unknown compounds detected in these metabolomic studies staggering. There is so much still to be discovered about our basic biochemistry. Not sure if I find this more encouraging or more discouraging.

 

[Hutan]

Yeah, good comments.
A bigger sample might have found metabolites that are significantly different at baseline?
Maybe the ME/CFS averages are being confounded by people in the sample who don't have ME/CFS?
A combination of those two things?
I don't know.
Yes, someone give Maureen and her team money please.

 

[Sean]

Anybody have any idea of the amount of money needed to do a large replication of this study?

 

[livinglighter]

How much do charities pay towards research? Can patients laise with charities to request the kind of studies we would like more funding to go towards?

 

[Hoopoe]

A failure to excrete toxic metabolic byproducts generated during exertion would fit with the poisoned feeling that many patients can relate to.

The assay used in this study doesn't detect small molecules. So there could be an accumulation that's not visible in the analysis.

(it's also showing what is being excreted, not what is potentially accumulating)

 

[mariovitali]

A failure to excrete toxic metabolic byproducts generated during exertion would fit with the poisoned feeling that many patients can relate to.

The assay used in this study doesn't detect small molecules. So there could be an accumulation that's not visible in the analysis.

So which organs are responsible to excrete toxic metabolic products (ie detoxification?)? That's right, the main organ is the Liver.

 

[Hoopoe]

So which organs are responsible to excrete toxic metabolic products (ie detoxification?)? That's right, the main organ is the Liver.

The liver being the hub of metabolism in the body does seem interesting in this context of a lack of response to exercise in many metabolic pathways.

 

[mariovitali]

Ultimately we have to know what has been working and what not. In an email sent to Professor Hanson on April 24th 2018 , I explained to her about the possibility that what we are dealing with is some kind of liver dysfunction, along with proposed mechanisms in a document that discussed detoxification, urea cycle , bile acid metabolism etc etc . Professor Hanson did in fact replied saying that "this information is quite interesting" and asked me what where the input data used in order to support the findings of the AI framework (which of course makes sense).

Here is my answer to her :



I will also post this on Twitter, the next things we should be looking at are the role of flavoproteins and sulfation and of course detoxification. I sure hope this does not take another 5 years to find.

 

[Hoopoe]

The other organ that seems like it could be relevant here would be the kidneys.

 

[butter.]

Ultimately we have to know what has been working and what not. In an email sent to Professor Hanson on April 24th 2018 , I explained to her about the possibility that what we are dealing with is some kind of liver dysfunction, along with proposed mechanisms in a document that discussed detoxification, urea cycle , bile acid metabolism etc etc . Professor Hanson did in fact replied saying that "this information is quite interesting" and asked me what where the input data used in order to support the findings of the AI framework (which of course makes sense).

Here is my answer to her :

View attachment 19032

I will also post this on Twitter, the next things we should be looking at are the role of flavoproteins and sulfation and of course detoxification. I sure hope this does not take another 5 years to find.

View attachment 19033

While I agree that particular end organs might be of greater importance than others in ME/CFS, I think we should think more in terms of specific organelles in specific cell types, rather than whole organs.

All (?) significant irregularities found in various metabollc studies can potentially be understood as mitochondrial and/or perixosomal dysfunction.

 

[Jaybee00]

Can patients laise with charities to request the kind of studies we would like more funding to go towards?

Of course. I do this with my donations to OMF and they acknowledge my requests.

 

[mariovitali]

While I agree that particular end organs might be of greater importance than others in ME/CFS, I think we should think more in terms of specific organelles in specific cell types, rather than whole organs.

All (?) significant irregularities found in various metabollc studies can potentially be understood as mitochondrial and/or perixosomal dysfunction

True, however we now have Prusty (a virologist) and Karl Morten suggesting "there may be something going on with the liver".

In the last papers of Hanson there are mentions on Liver disease and liver function.

Yes, it could be the kidneys but the major metabolic organ is the Liver. That is a fact. We fall into the trap believing that normal liver enzymes mean that there is no problem with the Liver. This is wrong.

 

[Hoopoe]

Acyl glycine fatty acid metabolites were the only compounds that stood out in ME/CFS patients.

Acyl glycines are altered specifically in disorders of branched-chain amino acid metabolism and fatty acid β-oxidation. The mitochondrial acyltransferase, glycine N-acylase, is expressed in liver and kidney.

Acyl glycine fatty acid metabolites are the only compounds that were found in urine at significantly different concentrations in ME/CFS vs. controls at a single time point (24 h post-exercise) and a different acyl glycine compound, 3-hydroxybutyroylglycine, had a significant negative correlation in the ME/CFS patients when correlating U3/U1 with P3/P1 (Figures 5 and 9). Additionally, cis-3,4-methyleneheptanoylglycine was changing
differently during exercise recovery in the ME/CFS patients vs. controls (LMM, Supplementary Figure S2). While acyl glycine metabolism is not one of the subpathways that was significantly increased post-exercise in the controls alone, it was significantly different in the ME/CFS patients vs. controls both at the 24 h post-exercise time point and when analyzing the difference in the post-exercise/baseline ratios (Figure 3). Urinary excretion of particular acyl glycines is also altered by disorders linked to fatty acid β-oxidation in the mitochondria, including medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD)deficiency [54]

I'm a carrier of MCAD deficiency. Maybe this acts as predisposing factor.

 

[mariovitali]

Thank you for sharing this regarding MCAD. Of course, it could all be a coincidence or even confirmation bias from my side (regarding the Liver) but i found the following :

When you don't have enough of the MCAD enzyme in your body, certain fats called medium-chain fatty acids can't be broken down and converted to energy. This results in hypoglycemia and low energy. Also, fatty acids can build up in body tissues and cause damage to the liver and brain.

https://www.mayoclinic.org/diseases-conditions/mcad-deficiency/symptoms-causes/syc-20353745

 

[FMMM1]

Just searched the paper for "24334" and noticed that it's related to "fatty acid metabolism"*. I think I recall Chris Armstrong highlighted differences re fatty acids - correct?

"Out of those four metabolites, only X-24334 is also changing significantly differently over time between controls and ME/CFS patients in the LMM and is increased after exercise in the control group."
Makes X-24334 even more interesting?

I wonder if GWAS will help to understand these findings? E.g. selecting using X-24334, and then doing GWAS, may provide clues re what the cause of the difference. Also, whole genome sequences may provide evidence of rare mutations i.e. indicating the (general) cause of this.

*Extract from paper:
"2.5. Acyl Glycines Have Lower Concentrations in the Urine of ME/CFS Patients Compared to Controls 24 Hours Post-Exercise The four metabolites found at significantly lower concentrations in ME/CFS vs. control subjects at the post-exercise time point in the univariate LMM analyses (q < 0.1) were 3-hydroxyoctanoylglycine, hexanoylglycine (C6), 2-octenoylglycine, and unknown X– 24334. The three known compounds are all in the Metabolon® subpathway acyl glycine fatty acid metabolism. The heatmap in Figure 5A shows the osmolality-normalized concentration post-exercise for these metabolites for every subject. Using agglomerative hierarchical clustering, the subjects clustered into three groups: (1) six control subjects, (2) two control subjects and one ME/CFS subject, and (3) the remaining nine ME/CFS subjects. The cluster of control subjects predominantly shows higher concentrations for all four metabolites while the cluster of ME/CFS subjects shows lower concentrations for all four metabolites. The small cluster with subjects from both groups shows intermediate values. The boxplots in Figure 5B demonstrate the minimal amount of overlap between the ME/CFS and control groups for these metabolites. Out of those four metabolites, only X-24334 is also changing significantly differently over time between controls and ME/CFS patients in the LMM and is increased after exercise in the control group."

 

[Hutan]

https://mdpi.altmetric.com/details/142429311

The countries that have people tweeting about the study include Peru, Japan, El Salvador and Croatia. The attention rating is "in the top 5% of all research outputs scored by Altimetric".


On MCAD, many of the symptoms sound familiar. However, MCAD is characterised by high levels of the acylglycines in urine:
Medium-chain acyl-CoA dehydrogenase deficiency. Diagnosis by stable-isotope dilution measurement of urinary n-hexanoylglycine and 3-phenylpropionylglycine

The levels of urinary hexanoylglycine and phenylpropionylglycine were significantly increased in all samples from the patients with MCAD deficiency, clearly distinguishing them from both groups of controls.

Hexanoylglycine is one of the four urinary compounds that differed from healthy controls 24 hours after exercise in the women with ME/CFS, but the levels of the four compounds were lower than those in the healthy controls.

 

[TiredSam]

The findings may be the beginning of something important but I think we should not lose sight of just how different it would be for a patient to go through a research procedure from a healthy control. When I started out on the wards I did a project looking at cortisol levels and they went up high when patients were admitted to the ward. I doubt they would have done for healthy subjects.

This is not an issue of 'psychological overlay'. It is just that the whole scene for a patient making an effort to contribute to a project relevant to there illness is going to be quite different from some odd member of staff or friend of a friend who has agreed to be a normal guinea pig.

Slight shifts in what is in one's mind can change eating, drinking and sleeping patterns. Patients might be better prepared physiologically from having raised their cortisol a bit beforehand - and so on.

About a year before I considered myself an ME sufferer, or had even heard of it, I started noticing that sport didn't seem to do anything for me any more (I had always done a lot of sport, enjoyed it and felt great afterwards). No more healthy glow after a day's cycling or a workout in the fitness studio. This was before ME affected my day-to-day life, it was just something I noticed with sport. I cycled with my two oldest boys from our house in Germany to my parents in England, 17 days, 1,500 km. I was really looking forward to getting and feeling fit that summer. But when we got there, nothing, I just felt like a tired old man. On one day of the trip I was suddenly and unaccountably more tired than I had ever been in my life, and just wanted to lie down in the road and have someone throw a blanket over me, but I kept going and it passed.

That autumn I was in the fitness studio for my regular workout when I felt a stabbing pain in my kidneys, just about managed to crawl home, and lay on the sofa for a few days. So began my ME journey about 10 years ago.

So I was a non-ME sufferer who stopped feeling the same after sport. I wasn't going through a research procedure or even aware that I had an illness or was part of an experiment.

 

[bobbler]

Intriguing study.

[My bold] So they want to expand their pilot study but don't currently have the funding to do it? Or am I misreading between the lines? Someone, anyone, please give this team a big pot of money!

There's one thing that's tying my brain into knots with studies like this one which point towards a failure of ME cells to mount an appropriate response to exertion. Why don't these studies show any difference at rest/baseline? We're most categorically ill all of the time, albeit more severely with PEM - so surely something must be measurably different already at baseline. Why does this not show up in the metobolomics?

Other studies have led to hypotheses that ME cells are stuck in a cell danger response or that they're exhausted (immune cells) or that they're running at full capacity and with all compensatory mechanisms switched on already at rest, or all of the above, and that the cells therefore are unable to respond to further demand. Which would fit the findings here that ME cells simply do nothing in response to exercise. But I would expect all of the hypotheses to impact metabolism at baseline as well as after exertion. So why do our baseline metabolomes look so seemingly normal? What are we missing? Something the tests aren't measuring? Yet they do throw up all manner of unidentified compounds. Or are the sort of pwME who participate in CPET studies so mild they're actually as well as the general population between episodes of PEM? That sounds unlikely to me.

On a different note, I find the sheer number of unknown compounds detected in these metabolomic studies staggering. There is so much still to be discovered about our basic biochemistry. Not sure if I find this more encouraging or more discouraging.

If this turns out to have potential even only in some areas it is pretty exciting - tho it takes my mind trying to bend thinking about what could or couldn't work just from this one defined study as an example re: the possibilities, and what could or couldn't be answered, or done methodologically - it feels something like this could fill some interesting knowledge and method/measure gaps.

One thing that is key about this 'politically' is, I suppose, that they have begun comparing 'equal absolute exercise-based exertion' in an acute sense (if you were a layperson reading this literally as the only exertion being the CPET both groups are both doing). So that we don't have any cart before horse 'less metabolites = maybe if they did more exertion to have these all would be great' stuff. And the fact the controls were sedentary with regards this issue.

It is interesting when you think of the methods and how everything needs to be done 'comparatively' (whether a change or a difference between groups), plus of course the broader timescales vs e.g. measuring HR or Vo2, and from a funding vs 'all the things you could do' and how you approach demonstrating it being worthy of further funding with a small pot to do one demonstration first up which angle you/one/I might have picked.

This to me doesn't seem like a bad starting point when you imagine the conundrum of all that might be involved with choosing one's 'project'. And yes then come in the whys, hows, and details and so on if more funding follows. Which mightn't have happened if you didn't pick well with that first one

Defining what 'rest' means and 'rested' is hard nevermind achieving it across an ME sample with heterogenous triggers, sensitivities, severities, cycles and so on when you've got PEM involved is perhaps a heck of a task. Whole thread there probably (hope it gets there one day).

I hope I'm not being naive in thinking that the urine aspect of this might be a positive because of the potential for this to be more 'in situ' long-term, allowing for at-home but also - if we can ever find a way for people to accept the 'same isn't equal' issue of ME/CFS re: methods - for instructions/process to be based on 'fit into your normal routine and take at the point when [e.g. you have just had a 'mega-rest' and feel at most rested, and then another measure after taking a shower]. And sort of combine with the 'heart rate in the home' type idea. It feels like the only chance of catching 'rest' as we'd get close to defining it (even using the broadbrush 'what most people think is a restful activity is actually an exertion even for milder PwME')?

BUT of course I don't know from these results whether we'll all be thrown by the absence of whatever being the finding. Or whether it is delay and so on. I like the fact that perhaps 24/4/6hr total urine collection (and could that potentially be done for e.g. a week follow-up) could be used to not add in unnatural exertion for PwME by prescribing precise timings whilst also not risking missing something due to a snapshot.

Then you have the need for someone who is a master at explaining methodologies to work out, if this still needs comparators, how to note that this is a good methodology vs the tendency for someone to instinctively think 'same conditions' involves having all of your sample doing the same thing at the same time etc.