Insights into ME/CFS phenotypes through comprehensive metabolomics (Lipkin, Hornig)

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Insights into myalgic encephalomyelitis/chronic fatigue syndrome phenotypes through comprehensive metabolomics
Scientific Reportsvolume 8, Article number: 10056 (2018) | Download Citation

Abstract
The pathogenesis of ME/CFS, a disease characterized by fatigue, cognitive dysfunction, sleep disturbances, orthostatic intolerance, fever, irritable bowel syndrome (IBS), and lymphadenopathy, is poorly understood. We report biomarker discovery and topological analysis of plasma metabolomic, fecal bacterial metagenomic, and clinical data from 50 ME/CFS patients and 50 healthy controls.

We confirm reports of altered plasma levels of choline, carnitine and complex lipid metabolites and demonstrate that patients with ME/CFS and IBS have increased plasma levels of ceramide. Integration of fecal metagenomic and plasma metabolomic data resulted in a stronger predictive model of ME/CFS (cross-validated AUC = 0.836) than either metagenomic (cross-validated AUC = 0.745) or metabolomic (cross-validated AUC = 0.820) analysis alone. Our findings may provide insights into the pathogenesis of ME/CFS and its subtypes and suggest pathways for the development of diagnostic and therapeutic strategies.
 
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FULL TEXT https://www.nature.com/articles/s41598-018-28477-9

Question: Is this finally that analysis that we've been hearing for years Lipkin would do when he had the money? I reckon yes - these samples are four years old!

Also a note: the title to this thread omits a LOT of impressive names who are listed as authors on this paper. Apologies to them.

Here's a few key excerpts:

Lipkin et al said:
Here we report targeted and untargeted analyses of 562 molecules representing primary metabolites, biogenic amines, lipid complexes and oxylipins in plasma of ME/CFS patients and controls. We also describe linkage of the resulting metabolomic data to a fecal metagenomic dataset and clinical data. As in our previously reported metagenomic analyses, we found that metabolomic profiles differ not only between ME/CFS patients and controls but also between ME/CFS patients who do or do not have IBS.
...


Subjects included 50 ME/CFS cases who met the criteria15,16 for ME/CFS and 50 matched healthy controls recruited at four sites across the United States (New York, NY; Salt Lake City, UT; Incline Village, NV; and Miami, FL). Subject demographics are shown in Table 1. The same subjects were enrolled as in our previous study14. Cases included 41 female and 9 male ME/CFS patients (mean age 51.1 years; standard error of the mean [SEM] 1.6). Controls included 41 female and 9 male subjects (mean age 51.3 years; SEM 1.6). All case and control samples were collected between June 22, 2014 and October 27, 2014. Irritable bowel syndrome (IBS) was diagnosed in 24 of the 50 ME/CFS patients (48%). One of the 50 control subjects reported a diagnosis of IBS.

...

Among the top plasma biomarkers differentiating ME/CFS patients from controls were decreased levels of betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM), and increased levels of triglycerides (TG), α-N-phenylacetyl-glutamine, ε-caprolactam and urobilin (Table S2). Set enrichment analysis of the results of logistic regression models revealed that ME/CFS subjects had reduced levels of PCs and dysregulation of the choline-carnitine pathway.

...

In ME/CFS with IBS, 5-methylthioadenosine (a metabolite derived from S-adenosylmethionine as a by-product of polyamine biosynthesis that can be toxic to mammalian cells21) was associated with functional impairment. Ceramides were associated with increased physical fatigue (Table S4). Decreased γ-butyrobetaine correlated with increased Faecalibacterium, a bacterium known to play an important role in the production of colonic butyrate, a by-product of fermentation with known beneficial impacts on intestinal barrier function and anti-inflammatory effects22,23.

...
The Naviaux group previously reported decreased plasma levels of SM, ceramides and PLs with the exception of PC 18:1 and PC 22:6, which were increased8. Tomic et al. reported increased plasma levels of TGs26. Germain et al. reported metabolomic biomarkers of disturbed amino acid, energy, sugar and fatty acid metabolism in ME/CFS patients13. Neither group examined the impact of IBS on metabolomic profiles in ME/CFS. Our analysis confirmed decreased levels of phospholipids and SM, and increased levels of TGs, but differed with respect to specific compounds. We did not find case-control differences in levels of PC 18:1 and PC 22:6. Nor did we find a consistent decrease in ceramide levels. Whereas ME/CFS without IBS had decreased levels of ceramides d43:1 and d42:1, ME/CFS with IBS had increased levels of six ceramide species: d36:1, d40:0, d42:0, d 34:1, d38:1 and d40:1.

Ceramide is a waxy lipid implicated in suppression of electron transport, insulin and leptin resistance and apoptosis27,28. Previous studies reported that ceramides might be involved in the pathology of IBS and metabolic disorders29,30. Increased levels of lipopolysaccharides (LPS) associated with an altered gut microbiome31 may trigger the activation of sphingomyelinases (SMAse) and the hydrolysis of SM to produce ceramides. Ceramides are toxic to many cell subtypes via the production of reactive oxygen species and may play a role in gut barrier dysfunction and increased gut permeability. Increased levels of ceramides were reported in mucosal samples from IBS patients29 as well as in plasma and tissue samples in diabetes, cardiomyopathy, insulin resistance, atherosclerosis and steatohepatitis. Blocking SMAse to decrease ceramide levels may be therapeutic in reducing inflammation32. Patients with ME/CFS and IBS also had higher plasma mannitol levels. We speculate that mannitol may increase permeability of both the gut mucosa and the blood-brain barrier resulting in trafficking of molecules such as cytokines and neurotransmitters that contribute to disease.

Similar to Armstrong and colleagues11, we found alterations in metabolites associated with mitochondrial energy metabolism
. Previous study reported significant decreases in TCA cycle metabolites related to energy metabolism in ME/CFS patients9. The reported dysfunction impacted carnitine metabolism and ATP/energy metabolism in the muscle of ME/CFS patients11. Carnitine is an important supplement that transfers acyl-CoA group into the mitochondrial matrix and participates in fatty acid β-oxidation (TCA cycle, ATP production and energy metabolism). Studies of carnitine levels in serum and plasma in ME/CFS have been inconclusive, with some groups reporting reductions whereas others find normal levels33,34. In an open label study of 30 patients, acetyl-carnitine supplements were reported to improve fatigue and cognitive function in up to 59% of patients with ME/CFS35. In our study (Table 2), compounds in the choline-carnitine pathway were decreased in ME/CFS patients regardless of their IBS status.

Our results are consistent with earlier reports that suggest that metabolites linked to lipid and energy metabolism are affected in ME/CFS. They extend earlier work by demonstrating that ME/CFS subjects with IBS co-morbidity have a distinct metabolomic profile compared to subjects without IBS and controls.

Many ME/CFS patients in our cohort take vitamin B supplements (26/50, 52%) that have the potential to increase levels of pantothenic acid. Use of vitamin B supplements was associated with higher levels of pantothenic acid and lower fatigue scores (data not shown). However, the numbers of samples were not sufficient to test for a significant relationship.
 
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percyval577

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Ive just read the article, a bit complicate written for me. S.3:

"Among the top plasma biomarkers differentiating ME/CFS patients from controls were

decreased levels of
betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM),

and increased levels of
triglycerides (TG), alpha-N-phenylacetyl-glutamine, epsilon-caprolactam and urobilin ..."
Also increased (table2) is phosphatidylethanolamine (PE).

They could (somehow) come to the result of a dysregulation of the choline-carnetine pathway, as they say on S.3.



S. 5-6 (results) shows up correlations with fecal bacteria, including "sympton severity".
S. 6: (discussion). "Plasma levels of 5-MT, a compound related to tryptophan, serotonin and melatonin metabolism, were decreased..." but their data were due to medications of the patients not sufficient.


S. 7: "ME/CFS is a heterogeneous disorder."
 
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Re-read it. Six thoughts

1. They confirm some of the previous metabolomic findings - ceramides and phospholipids.
2. They find (yet) another subgroup that can confound metabolomic findings - having IBS.
3. Gut Bacteria matter - they can help identify the disease.
4. They include 9 men in the 50 patients and 9 in the 50 controls, and this adds no statistical power on the male side, while diminishing statistical power on the female side. Not sure it's worth it.
5. A treatment idea: They find some possible reasons to take b-vitamins.
6. These topological maps are interesting to look at but I can't get much out of them in terms of actionable ideas, beyond the fact that the connections look mostly non-random (i.e. There's *something* there.)

41598_2018_28477_Fig3_HTML.jpg
Topological data analysis (TDA) revealed altered metabolomic and metagenomic profiles in ME/CFS. The color scheme represents the strength of association with ME/CFS diagnosis (white: strongly associated with control, red: strongly associated with ME/CFS). Each node in a network comprises 1 or more subject(s) who share variables in multiple dimensions. Lines connect network nodes that contain shared variables and subjects. Unlike traditional network models wherein each node reflects only a single sample, the size of a node in the topological network is proportional to the number of variables with a similar profile. (A), (B) and (C) integrate plasma metabolomic, fecal metagenomic and plasma immune profiles, and symptom severity scores using the Jaccard metric to define multidimensional subgroups. Irrespective of the lenses used [(A) neighborhood lenses NL1 and NL2, (B) MDS coordinate 1 and 2, and (C) metric PCA 1 and 2], ME/CFS and control samples formed distinct networks. ME/CFS and control samples also formed distinct networks in TDA based on either fecal bacterial relative abundance or plasma metabolomic data in isolation using a variance normalized Euclidean distance metric with neighborhood lenses (NL1 and NL2). Fecal bacterial relative abundance features (D) were stronger drivers of the network distinction than plasma metabolomic features (E).
 

Neunistiva

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They find (yet) another subgroup that can confound metabolomic findings - having IBS.
I'm not sure if I would call this a subgroup because I had ME/CFS for 6 years and was already mostly bedridden by the time I developed IBS. I accidentally drank a yogurt that has expired and a few hours later I developed IBS that hasn't stopped to this day, 3 years later.

It wouldn't make sense that I would be categorized into different subgroups just because I got tested pre-yogurt or post-yogurt.

So I wonder, instead of a being a distinct subgroup, if we don't all just have a predisposition to develop IBS and whether we have it or not just depends on whether we ran into a trigger or not, and then our metabolic profile changes (and probably gut microbiome).

Did anyone else develop IBS or stop having IBS while having ME/CFS?
 

Learner1

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Among the top plasma biomarkers differentiating ME/CFS patients from controls were decreased levels of betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM), and increased levels of triglycerides (TG), α-N-phenylacetyl-glutamine, ε-caprolactam and urobilin (Table S2). Set enrichment analysis of the results of logistic regression models revealed that ME/CFS subjects had reduced levels of PCs and dysregulation of the choline-carnitine pathway.
Also increased (table2) is phosphatidylethanolamine (PE).
PE would be increased because its not converting to PC? This might affect cell and mitichindrial membranes.

https://en.m.wikipedia.org/wiki/Phosphatidylethanolamine_N-methyltransferase

I had a couple of PC IVs recently and felt markedly better after each.
 
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percyval577

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I don´t know, I asked myself though.
Maybe the body wants or wants not to build membranes?
Accordingly, whats about the free TG´s? whatfor?

Or can it be understood by a lactate-mechanism?
 
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Learner1

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Part of it could be that they're destroyed faster than they can be made, and also that the making of them is slowed down. At least that's the theory my doctors are working with based on some other data points.

This might have some clues, but it's a little
dense:

https://bmccellbiol.biomedcentral.com/articles/10.1186/s12860-014-0043-3

Lipids are in a constant flux and are continuously converted into each other. Within cells they can move within membranes and between different cellular compartments. Furthermore, lipids are exchanged between different tissues. Extracellularly, the bulk of lipids is transported in lipoproteins. These lipoproteins are soluble complexes of proteins (apolipoproteins) and lipids that are transported in the circulation of vertebrates and insects and that are synthesized in the liver and intestine.
...
The major neutral lipid, triacylglycerol (TAG), is secreted from the liver and intestine...
 

Diwi9

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I am also curious if the results are skewed in the IBS group as so many of us take on specific diets to help control symptoms. I don't recall any mention of this in the paper.
 

percyval577

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To me the paper does not look like an attempt to understand anything. It´s even written somehow, well confuse might be said to much.

Only a lot of different people if Ive grasped it right have paid a lot of effort to the huge amount of investigations and findings announced there, and that´s it.

So, the findings I would guess to be right (but to be independtly confirmed of course), and any further guesses are still be to figured out.

Also, one must say that the paper does not dig into manny (possible) subgroups, whatever special there might occure.
But I guess the databank the findings are steert to can reveal then at some point.
 

percyval577

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Part of it could be that they're destroyed faster than they can be made, and also that the making of them is slowed down. At least that's the theory my doctors are working with based on some other data points.
Could be, but to be honest it seems to me a baby-step-theory.
Most often doctors are not the ones who are able to come up with theories and would think in a new direction on their own. And the next patient is already waiting (anyway).
I know there are a lot of very engaged doctors, doing a lot for the patients even not being paid for. I have met doctors when they go home in the evenning. But all effort they spend will not enable them to come up with a nice theory (new guess).
 
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Does this fall under the theory that the pyruvate dehydrogenase enzyme is inhibited? This would result in decreased production of acetyl coenzyme A and acetyl CoA is needed to produce acetylcholine. Without acetylcholine, the nervous system wouldn't be able to function, so it's very vital to life. It only recently occurred to me that the cell membranes act as a reservoir of choline to make acetylcholine. Choline can also be made in the liver from methionine. Carnitine is also made from methionine (plus lysine). Lysine degrades to acetyl CoA and is a ketogenic amino acid that would likely get burned for energy if fat and carbs cannot be burned.

I'm trying to put the pieces together, but I feel like I'm not quite getting the big picture. My fatigue and brain fog seem to be caused by lack of acetylcholine (central nervous system fatigue/central command fatigue/bonking/whatever you want to call it). However, just taking acetylcholine precursors/cofactors doesn't entirely fix me, just almost entirely. The choline needs to be acetylated by acetyl CoA before it becomes acetylcholine, and I think that's the rate-limiter for me. I believe acetyl CoA is made from lysine and cysteine (which would be derived from methionine if sufficient cysteine is not gotten through the diet). I mean technically every protein made in the human body would require methionine as it is always the start codon. Lysine is a pretty high order amino acid too; it's very vital to human health.

So low lysine would result in recurrent viral infections (which I had when I got sick). Low methionine could conceivably cause lack of monoamine neurotransmitter production, lack of carnitine, lack of glutathione... (all of which I seem to have). And the low acetylcholine could cause gut problems/dysbiosis. Could it be so simple as a choline/amino acid deficiency for some of us? I can see that my hair and skin are depleted of something, likely cysteine and/or lysine. I could be way off track, but my response to these supplements (acetylcholine support, acetyl CoA support, phospholipid replacement therapy) suggests that I'm not. It's just so hard to tell if it's just a Band-Aid or if I'm discovering my root cause.

I guess that's a really long way of saying that I'm not surprised they found phospholipid/choline/carnitine aberrations.
 
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PE would be increased because its not converting to PC? This might affect cell and mitichindrial membranes.

https://en.m.wikipedia.org/wiki/Phosphatidylethanolamine_N-methyltransferase

I had a couple of PC IVs recently and felt markedly better after each.
Yes, I would love to know if this affects mitochondrial membranes, especially the inner mitochondrial membrane where the electron transport chain is. A leaky membrane here could result in a lot of oxidative stress?
 

Learner1

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So low lysine would result in recurrent viral infections (which I had when I got sick). Low methionine could conceivably cause lack of monoamine neurotransmitter production, lack of carnitine, lack of glutathione... (all of which I seem to have).
I've been in the same boat...we kept solving these amino acid problems til my doctor ordered 40g a day of a custom amino powder.

And the low acetylcholine could cause gut problems/dysbiosis. Could it be so simple as a choline/amino acid deficiency for some of us?
These are things that my doctors have been looking at. I think these may be a part of the puzzle, but not sure they'll solve the various immune issues.
I can see that my hair and skin are depleted of something, likely cysteine and/or lysine. I could be way off track, but my response to these supplements (acetylcholine support, acetyl CoA support, phospholipid replacement therapy) suggests that I'm not.
Wonder how Mestinon figures in here? What are you using to support acetyl CoA? Pantethine, BCAAs? And are you using oral or IV phosphatidyl choline, NT Factor, or??