Metabolomic Evidence for Peroxisomal Dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2022, Levine,Hornig,Lipkin et al

I clearly remember that Ron Davis some years ago said they were seeing high levels of lipids in cells in one of the experiments they did. Maybe in connection with cells being lighter?

Accumulation of lipids is something that can happen when the peroxisomes are not working well.

 

A blog looking at, in part, this study.

Peroxisomes, Mitochondria, And Fibrinoid Microclots: Two New Preprints.

https://www.meprecisely.com/2022/07...a-and-fibrinoid-microclots-two-new-preprints/

 

I have a low allele frequency mutation in the gene for the first step in the synthesis of plasmalogens. It could be a coincidence but it's also suspicious.

Also a mutation in a choline transporter that is involved in the synthesis of phospholipids and acetylcholine.

Affordable whole genome sequencing seems like a very powerful tool, if only we can improve our ability to interpret all the mutations that one can find with it. We need more data.

 

Cort’s take is here

https://www.healthrising.org/blog/2022/08/21/lipkin-chronic-fatigue-syndrome-puzzle/

 

If anyone has questions on this study, I can forward to my contact at Columbia. Probably better to forward as a batch versus one by one.

 

I have questions:

Do they think that plasmalogens could be a useful drug target? I noticed that there is some interest in treating other diseases with plasmalogen replacement.

Do they think carnitine supplement could be an effective treatment?

Do they have any thoughts on what could be causing this kind of problem?

 

I have read in two different articles that beta-adrenergic signalling is linked to peroxisome activity. People with a genetic disorder that impacts peroxisome formation have more beta-adrenergic receptors on their cells. In cell cultures, beta-adrenergic stimulation also reduces peroxisome gene expression.

So in a very superficial way, it looks like the peroxisome dysfunction might be a result of excessive beta-adrenergic signalling. But there is little information on this available. This stood out to me because a problem with beta-adrenergic signalling (due to autoimmunity) is one of the popular hypotheses on ME/CFS

 

Review article on peroxisome protein importing (paywalled) —

Import and quality control of peroxisomal proteins (2023)
Rudowitz, Markus; Erdmann, Ralf

Peroxisomes are involved in a multitude of metabolic and catabolic pathways, as well as the innate immune system. Their dysfunction is linked to severe peroxisome-specific diseases, as well as cancer and neurodegenerative diseases. To ensure the ability of peroxisomes to fulfill their many roles in the organism, more than 100 different proteins are post-translationally imported into the peroxisomal membrane and matrix, and their functionality must be closely monitored.

In this Review, we briefly discuss the import of peroxisomal membrane proteins, and we emphasize an updated view of both classical and alternative peroxisomal matrix protein import pathways. We highlight different quality control pathways that ensure the degradation of dysfunctional peroxisomal proteins. Finally, we compare peroxisomal matrix protein import with other systems that transport folded proteins across membranes, in particular the twinarginine translocation (Tat) system and the nuclear pore.

Link | PDF (Journal of Cell Science)

 

Interesting. The lipidomics I presented at IACFSME 2023 showed broadly elevated levels of lipids in lymphoblastoid cell lines generated from pwME.

 

I'm revisiting some metabolomics papers and thought I'd share some thoughts about this one.

I think it's potentially quite interesting. It's got a decently large cohort especially by ME research standards 101 pwME, 91 controls, seem fairly well matched in most areas, recruited from different sites, CCC criteria.

Statistics

They use fairly complicated statistics in this paper to determine significant metabolites. They use a Bayesian linear regression model where they also account for other features like age, ethnicity and so on.

I think their mixed effects linear model is a good thing as it allows you to see the variation in the data that is only coming from the difference between the ME and HC groups with the variation due to other features stripped away. I didn't see them report anywhere on whether there was an effect with age, ethnicity, size and so on. That would have been interesting to see. Of course you can't say for sure that they didn't try analyzing in different ways too to try and find the most compelling results.

Instead of simply comparing a p value they also have cut offs from a bayesian analysis of the data. Their criteria for significance is an FDR adjusted p < 0.15, a Bayes factor > 3 and 95% HDI not overlapping with 0. It's quite a complicated way to delineate interesting metabolites.

What the Bayes factor effectively means is 'how many times better does the alternative hypothesis fit the data than the null hypothesis' after a Bayesian analysis. A Bayes factor of 3 is generally interpreted as providing moderate evidence for your alternative hypothesis (that ME and HC have different levels of a given metabolite). The HDI is basically saying that there is a 95% chance that there is a difference between the groups (I've oversimplified a little bit there).

I'm not experienced enough in Bayesian statistics to know whether using all three of these metrics is really more powerful than just using a p-value, or whether practically it's basically saying the same thing 2 or 3 different ways. The cynic in me would say they've used an adjusted p-value cut off of 0.15 instead of 0.05 because they barely saw anything at the 0.05 thereshold. Then again, you can see for example in these two metabolites that they have similar adjusted p values (second from the right) but vastly different Bayes factors (right most column):



(A bayes factor of 178 would be interpreted as extreme evidence in favour of a difference between HC and ME for this metabolite).

 

Biology

This sounds interesting. It's interesting that all the reductions in membrane lipids they find are unsaturated (containing double bonds rather than completely saturated with hydrogens). That is matched by an increase of unsaturated (but not saturated) triglycerides. Triglycerides are the storage form of fatty acids and are stored in lipid droplets that are visible inside of the cell. So if as the authors argue there is a peroxisomal problem, and that affects synthesis of membrane lipids containing unsaturated fatty acids, maybe that would follow that you would have an accumulation of unsaturated triglycerides and lipid droplets.

Comparison to Naviaux's ME/CFS metabolites paper

As the authors say they replicate a lot of the same findings found in pwME from Naviaux et al 2016. The strongest signal in that paper was a reduction in the levels of various membrane lipids in particular sphingolipids (ceramides) and phospholipids. They don't however replicate Naviaux's findings of lower purine metabolites (uric acid, xanthine), or glutamate related metabolites (arginine, pyrroline 5-carboxylate). I think these findings are also potentially interesting, especially the glutamate ones (for some other reasons from other papers).

I agree that diet might be the most obvious source of variance. In this paper the participants are sampled after overnight fasting. The Naviaux paper only required a 3 hour fast before sampling. I also think that differing levels of exercise might affect the results. The patients may have had to travel to be sampled which might have involved exertion.

Another possibility is sample storage. Metabolites have vastly different concentrations and degradation rates, and they can also degrade into other metabolites. Glutamine can spontaneously deaminate to glutamate for example. So the way the samples have been stored and for how long matters.

Finally, they see see an increase in levels of alpha-ketoglutarate, the TCA cycle metabolite and 2-oxo acid version of glutamate. This is the opposite of what other metabolomics sources report. Wüst's team (Appelman et al 2024) recently reported a reduction in glutamate and alpha-ketoglutarate in the plasma from their muscle study for example. I wonder if changes in these molecules could be an exercise related effect.

 

I don't know if this means anything, but my alpha-ketoglutarate (urine) was at 1 (ref range 5-80) during 2 day post-exercise PEM.

 

Had a look in hanson urine study and it is lower relative to controls (0.7 of controls) but not significant p~0.3. Armstrong has also done some urine stuff but doesn't seem to have reported it.

 

This paper has been extended by Che, Lipkin and Fiehn here:

https://www.s4me.info/threads/bayes...ydges-che-lipkin-and-fiehn.27730/#post-496038

They bundle the results of their paper with Nagy-Szakal's paper and Naviaux's paper in a sort of Bayesian meta-analysis and the peroxisome pops out even more.

fulltext: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10535181/

We also found that only unsaturated triacylglycerides were upregulated in ME/CFS patients, but not saturated triacylglycerides. Such a finding points to a specific biochemical mechanism instead of simple explanations such as differences in the number or type of lipid-carrying lipoprotein particles. A further indication for this mechanism was a profound downregulation of unsaturated phospholipid ethers and plasmalogens (Figure 6 and Figure 7), which are exclusively produced by peroxisomes and which, hence, might be involved in the etiology of the disease [17]. Importantly, the results of the Bayesian analysis further strengthened the biological interpretation of peroxisome damage as an important factor underlying ME/CFS: very long chain polyunsaturated triacylglycerides were found at increased levels in ME/CFS subjects (Figure 6), pointing to a lack of oxidation in peroxisomes that exclusively perform this reaction (not mitochondria).

Lipids and membranes could be the bridge to the Hwang paper on endoplasmic reticulum stress and wasf3. Could the ER be perpetually stressed because a lipid imbalance in the membrane means it runs at reduced efficiency?

 

Peroxisomes help make plasmalogens. A plasmalogen shortage is one of the findings by Lipkin. Also one of the theories in this hypothesis paper from Canada: https://www.s4me.info/threads/chron...sis-addressing-the-overlap-2023-chaves.34083/

Plasmalogens are an intriguing idea to pursue because plasmalogen replacement therapy is established and it's a non-toxic supplement so collecting some anecdotal data should be straightforward.

I regret that the supplement itself has an animal-linked name that makes it sound suspicious rather than a scientific name that makes it sound serious, nevertheless a team of scientists from a good university have found it effective in raising blood levels of plasmalogens:

Shark liver oil supplementation enriches endogenous plasmalogens and reduces markers of dyslipidemia and inflammation

https://pubmed.ncbi.nlm.nih.gov/34146594/

 

A few recent papers relating to peroxisomes are —

The peroxisome: an update on mysteries 3.0 (2024, Histochemistry and Cell Biology)

Mitochondrial-derived vesicles in metabolism, disease, and aging (2024, Cell Metabolism)

Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network (2023, Nature Cell Biology)