Search for biomarkers in ME/CFS using Raman spectroscopy - ME Association (UK)

[Hip]

We need to ask one of the more scientific among us what 0 is the symbol for.

I think 0 cells (or 0 cells in some browsers) is an error in the text display, because in the full paper on Sci Hub, it reads as ρ0 cells.

The symbol ρ is the Greek letter rho, and ρ0 cells are apparently cells in which the mitochondrial DNA has been depleted (removed).

 

[boolybooly]

To be critical of this, ρ0 cells' accumulation of phenylalanine does not prove PWMEs PBMCs accumulate it in the same way. So it carries zero scientific weight re: mechanism and at most ρ0 cells act as another form of control showing that the Raman spectroscopic technique detects real phenomena.

 

[Murph]

If replicable it suggests CFS involves blood cells (PBMCs) importing phenylalanine, accumulating it internally while lowering its concentration in the plasma.

The $64k question is ... why? Also is it just PBMCs or are other cell types doing the same?

As to why, the paper suggests:

> Our results suggest that the patients’ cells could be accumulating more amino acids (e.g. phenylalanine) which results in a reduction in the fluids observed in other studies. This may be due to an induction of secondary rescue mechanisms to intracellularly accumulate more amino acids and maintain a normal ATP production in the metabolically dysfunctional patients’ cells.

i.e. cells are hogging aminos to make ATP. (but why only phenylalanine?) they point out that they are not sure.

Anyway, this was a pilot study, basically demonstrating that the technology - Raman spectroscopy - is one that could be useful in studying me/cfs mitochondria. It's another thing to put in the toolbox.

 

[pattismith]

Berti and colleagues [90] showed that bilateral administration of creatine or pyruvate into hippocampus significantly prevented the cognitive impairment triggered by Phe administration in rats in the open field apparatus, indicating that cognitive impairment found in phenylketonuric patients might be secondary to energy failure. Significant beneficial effect of creatine and pyruvate administration was confirmed by the prevention of adenylate kinase (EC # 2.7.4.3), mitochondrial and cytosolic CK activities impairment in cerebral cortex and hippocampus from pregnant and lactating rats receiving high Phe administration, corroborating the hypothesis of impaired energy metabolism on Phe neurotoxicity [91].

I just started calcium pyruvate + creatine today, and all I got for the moment is a big worsening of my tinnitus 1 h 30 after the intake...

 

[FMMM1]

As to why, the paper suggests:

> Our results suggest that the patients’ cells could be accumulating more amino acids (e.g. phenylalanine) which results in a reduction in the fluids observed in other studies. This may be due to an induction of secondary rescue mechanisms to intracellularly accumulate more amino acids and maintain a normal ATP production in the metabolically dysfunctional patients’ cells.

i.e. cells are hogging aminos to make ATP. (but why only phenylalanine?) they point out that they are not sure.

Anyway, this was a pilot study, basically demonstrating that the technology - Raman spectroscopy - is one that could be useful in studying me/cfs mitochondria. It's another thing to put in the toolbox.

I once came across something similar to this. I.e. using infrared spectroscopy to determine calorific value of food. If you measure the calorific value of foods (using conventional time consuming methods), and the infrared spectrum of the same sample, then you can derive a relationship and then determine the calorific value using infrared spectroscopy (much quicker/cheaper method).

This could potentially be used in the same way. E.g. use the Seahorse analyser/nano needle to generate reference data and then check this against the Raman spectroscopy data. Ron Davis reviewed a range of techniques i.e. as potential biological diagnostic tests for ME/CFS [Invest in ME Conference - June 2018]. Possibly Raman spectroscopy could be added to the list of potential biological diagnostic tests for ME/CFS.

When are we going to get one of these tests delivered i.e. as a biological diagnostic test? How can we encourage the funding bodies e.g.Horizon 2020 [European Union science fund], or NIH, to fund the delivery of a biological diagnostic test?

Some further thoughts:
Ron Davis previously commented that ME/CFS looked a lot like diabetes [OMF talk - YouTube]. I assume that this supports that view i.e. mitochondrial dysfunction. Also, this may provide information regarding the nature of the metabolic defect --- what's accumulating/what's likely to be the cause/blockage. Also, bear in mind that Fluge and Mella found that the effect tracks with the plasma. Interesting to see if this technique [Raman spectroscopy] would demonstrate something similar. This looks like a positive development but is it reversible i.e. downstream effect of a "metabolic trap"?

 

[FMMM1]

I assume that the idea i

I think 0 cells (or 0 cells in some browsers) is an error in the text display, because in the full paper on Sci Hub, it reads as ρ0 cells.

The symbol ρ is the Greek letter rho, and ρ0 cells are apparently cells in which the mitochondrial DNA has been depleted (removed).

I assume that ρ0 cells are included in order to give them something to compare the results in ME/CFS cells to. If you check out my other post you'll see that normally there's a "reference method", i.e. the current technique, and the spectroscopy technique can be compared to this. In this case there is no current reference method (biological diagnostic test). They've picked a mitochondrial reference, i.e. cell with no mitochondrial DNA, and they're saying ME/CFS looks like a mitochondrial disease. Ron Davis said something similar in a You Tube talk (OMF site). Good news story here i.e. this looks like promising research.

 

[FMMM1]

If phenylalanine was a cause of symptoms, then consuming aspartame should increase symptoms severity. I've had aspartame and notice no such effects. Of course, taking normal quantities of aspartame may not be enough to increase levels in cerebral cells significantly.

Not sure what's going on here. Here's an extract from Chris Armstrong's 2015 paper*
"3.2 Altered blood metabolites in patients with ME/CFS
The absolute concentrations of six blood metabolites from the 1H NMR analysis were significantly altered. Glucose levels were increased whereas acetate, glutamate, hypoxanthine, lactate, and phenylalanine were decreased in ME/CFS patients ---these correlations, paired with a decrease of amino acid concentrations, implicate an increasing utilization of amino acids as a source of energy production".
So Chris reckoned the low concentrations of phenylalanine in blood plasma were due to the body switching to use phenylalanine as an energy source in ME/CFS.

So is the high level of phenylalanine inside the cell (found in this study) due to it being absorbed into the cell as an energy source?

Could you/should you supplement with phenylalanine i.e. as an energy source?

Anyone got a link to the whole paper?

*https://minerva-access.unimelb.edu....g Metabolomics CFS.pdf?sequence=5&isAllowed=y

 

[FMMM1]

The authors of the study hypothesize that as phenylalanine was low in patient's sera and urine in previous studies,
the elevated intracellular concentration of phenylalanine found here 'may be due to a secondary rescue mechanism (...) to maintain a normal ATP production in the metabolically dysfunctional patients' cells' (page 9 of the accepted manuscript).

A bigger study to confirm their findings is 'under preparation' (p. 10).

Interesting Chris Armstrong reckons phenylalanine was being used as fuel; which I assume is ATP production [https://minerva-access.unimelb.edu.au/bitstream/handle/11343/121955/2015 Armstrong Metabolomics CFS.pdf?sequence=5&isAllowed=y]. Search for "phenylalanine" and you'll find that he proposes that it's low as it's used for energy production.

Winging it here. Regarding the production of ATP from phenylalanine. I wonder if they have done/could do/plan to do a stable isotope/mass spectrometry study to see if this is correct i.e. test whether phenylalanine is converted to ATP. Phair's currently doing stuff along these lines i.e. a stable isotope/mass spectrometry study.
These authors state that: "As mitochondria are the primary energy power plant of most eukaryotic cells and supply the cell with metabolic energy in the form of ATP".
I assume that this is the same as Chris Armstrong.

Could you direct me to a copy of the full paper?

 

[Lisa108]

Could you direct me to a copy of the full paper?

Hi @FMMM1, do you mean the paper from the beginning of this thread?
sci hub link
Sorry if I misunderstood, bad brainfog day...

 

[FMMM1]

Hi @FMMM1, do you mean the paper from the beginning of this thread?
sci hub link
Sorry if I misunderstood, bad brainfog day...

Thanks.This is the paper I was looking for.

 

[Wishful]

I doubt that a small amount of aspartame is going to boost ATP production noticeably, so it's not of much value as a test for that hypothesis. I admit that home experiments are a limited value for disproving hypotheses, due to absorption, processing, or transport dysfunctions of a primary factor and cofactors.

 

[FMMM1]

To be critical of this, ρ0 cells' accumulation of phenylalanine does not prove PWMEs PBMCs accumulate it in the same way. So it carries zero scientific weight re: mechanism and at most ρ0 cells act as another form of control showing that the Raman spectroscopic technique detects real phenomena.

I need to read the paper and even then I reckon I'll struggle to understand this.

Phenylalanine can I assume be identified in a Raman spectrum. I.e. the elevated peak in the spectrum, in people with ME/CFS, is phenylalanine.

Chris Armstrong found that phenylalanine was low in blood plasma and proposed that it was being used for energy production (ATP production) [https://minerva-access.unimelb.edu.... Metabolomics CFS.pdf?sequence=5&isAllowed=y]. Others have to my knowledge also found this [Fluge and Mella].

I don't accept that this "carries zero scientific weight re: mechanism".

The "total accuracy of 98.1% was achieved based on 206 Raman spectra" would also appear to be at odd with this statement.

My knowledge of science is limited but I don't see a whole lot to concern me at this point. It's very confusing but I think that is due to the complexity of the subject and the limitation of the tools.

I hope to see the larger study soon.

 

[FMMM1]

As to why, the paper suggests:

> Our results suggest that the patients’ cells could be accumulating more amino acids (e.g. phenylalanine) which results in a reduction in the fluids observed in other studies. This may be due to an induction of secondary rescue mechanisms to intracellularly accumulate more amino acids and maintain a normal ATP production in the metabolically dysfunctional patients’ cells.

i.e. cells are hogging aminos to make ATP. (but why only phenylalanine?) they point out that they are not sure.

Anyway, this was a pilot study, basically demonstrating that the technology - Raman spectroscopy - is one that could be useful in studying me/cfs mitochondria. It's another thing to put in the toolbox.

Regarding the authors selection of phenylalanine, if you search the manuscript for "phenylalanine" you'll see a bunch of reasons/explanations e.g.:
"The most prominent feature was observed at 1003 cm-1, which can be assigned to aromatic ring vibrations of phenylalanine." i.e. it jumps out of the spectrum
"All bands related to phenylalanine were higher in the ρ0 cells compared with the WT cells (Fig. 3A), which indicates an important role of the aromatic amino acid, phenylalanine, in the metabolism of cells with mitochondrial dysfunction." I think others found this before e.g. Chris Armstrong [2015] and others after Chris [Fluge, Mella etc].
"As phenylalanine alone might not be sufficient to characterize the pathomechanism, further biomarkers should be identified to simultaneously and more reliably identify mitochondrial dysfunction." They'll look at other compounds presumably including amino acids found to be low by Chris Armstrong etc.

I agree that this could be a useful tool.

As the authors say this has "the potential to be used as a diagnostic tool of CFS". Here's a few extracts:
"Both of the signature bands of phenylalanine were found to be significantly higher in the patients compared to the controls (p < 0.0001)." Odds ratio 1:10000; impressive.
"As one Raman spectrum can be obtained within seconds, one patient sample that consists of multiple spectra and multiple cells can be characterized and classified within a few minutes, which implies an enormous potential and feasibility in clinical practice."
"The combination of Raman biomarkers and classification models might lead to improvements in our understanding of CFS pathogenesis and have the potential to be used as a diagnostic tool of CFS."

 

[boolybooly]

I need to read the paper and even then I reckon I'll struggle to understand this.

Phenylalanine can I assume be identified in a Raman spectrum. I.e. the elevated peak in the spectrum, in people with ME/CFS, is phenylalanine.

Chris Armstrong found that phenylalanine was low in blood plasma and proposed that it was being used for energy production (ATP production) [https://minerva-access.unimelb.edu.au/bitstream/handle/11343/121955/2015 Armstrong Metabolomics CFS.pdf?sequence=5&isAllowed=y]. Others have to my knowledge also found this [Fluge and Mella].

I don't accept that this "carries zero scientific weight re: mechanism".

The "total accuracy of 98.1% was achieved based on 206 Raman spectra" would also appear to be at odd with this statement.

My knowledge of science is limited but I don't see a whole lot to concern me at this point. It's very confusing but I think that is due to the complexity of the subject and the limitation of the tools.

I hope to see the larger study soon.

Yes it is complicated and its doubly difficult to make sense of it all with a head full of brain fog.

I feel that we are really spectators here so no harm in chewing the fat I dont want to seem argumentative but would like to say why I said that to explain myself to you and anyone else reading.

Even the authors themselves state ...

As similar changes were observed in the ρ0 cell model with a known deficiency in the mitochondrial respiratory chain as well as in CFS patients, our results suggest that the increase in cellular phenylalanine may be related to mitochondrial/energetic dysfunction in both systems.

I would say that was a big "may " and tbh I consider it highly speculative and unsafe even as conjecture at this juncture, which is what I am trying to say, I am sorry but this is hype. If they want to hypothesise based on that idea then OK that is the next step but then they have to devise experiments to prove it and it ain't proved yet.

The problem with the ρ0 cells as a model is we dont even know if ME includes a metabolic block or trap yet. ρ0 cells are not derived from PWME, they have no mitochondrial DNA and are a kind of knockout mutant essentially and the similarity with ME cells is not proven so the comparison proves absolutely nothing about why PWME PBMCs accumulate phenylalanine.

We dont know why either cell type accumulates phenylalanine, is it because they are not burning it so it accumulates? Or is it because there is a regulating feedback mechanism which would reduce accumulation which is blocked so it just piles in, or is it because they are metabolically stressed and actively importing it to compensate via an active feedback mechanism? We simply dont know and it could be different reasons in the different cell types.

I would also comment that we dont know what this technique shows with other illnesses or stress states and it may be a common challenge response to accumulate phenylalanine.

For each one of these questions definitive controlled experiments are needed to get an answer and they havent been done so presuming that the accumulation of phenylalanine in both cell types is due to the same cause is a long shot and not safe at this point.

I agree the finding makes me feel very curious and maybe even hopeful that there is something worth looking at but my feeling is we should not get ahead of ourselves and make unsafe presumptions because as we have seen before, with things like XMRV, with bio-sciences presumptions can be turned on their head with one simple experiment going the opposite way from what you might presume, thus showing that reality is not what we believe it to be.

That is science and its why it helps because it reveals reality but it is also why we should be careful to understand the limits of our knowledge and try to be very logical about interpreting and designing experiments. That is why I say this shows us nothing about the reasons why PWME PBMCs accumulate phenylalanine but it does challenge us to find that out.

For full disclosure its a bit of a hobby horse of mine because I did a bioscience degree a long time ago and then I qualified as a biology teacher so I feel its my duty to point this kind of thing out but please don't hold it against me!

 

[FMMM1]

Yes it is complicated and its doubly difficult to make sense of it all with a head full of brain fog.

I feel that we are really spectators here so no harm in chewing the fat I dont want to seem argumentative but would like to say why I said that to explain myself to you and anyone else reading.

Even the authors themselves state ...



I would say that was a big "may " and tbh I consider it highly speculative and unsafe even as conjecture at this juncture, which is what I am trying to say, I am sorry but this is hype. If they want to hypothesise based on that idea then OK that is the next step but then they have to devise experiments to prove it and it ain't proved yet.

The problem with the ρ0 cells as a model is we dont even know if ME includes a metabolic block or trap yet. ρ0 cells are not derived from PWME, they have no mitochondrial DNA and are a kind of knockout mutant essentially and the similarity with ME cells is not proven so the comparison proves absolutely nothing about why PWME PBMCs accumulate phenylalanine.

We dont know why either cell type accumulates phenylalanine, is it because they are not burning it so it accumulates? Or is it because there is a regulating feedback mechanism which would reduce accumulation which is blocked so it just piles in, or is it because they are metabolically stressed and actively importing it to compensate via an active feedback mechanism? We simply dont know and it could be different reasons in the different cell types.

I would also comment that we dont know what this technique shows with other illnesses or stress states and it may be a common challenge response to accumulate phenylalanine.

For each one of these questions definitive controlled experiments are needed to get an answer and they havent been done so presuming that the accumulation of phenylalanine in both cell types is due to the same cause is a long shot and not safe at this point.

I agree the finding makes me feel very curious and maybe even hopeful that there is something worth looking at but my feeling is we should not get ahead of ourselves and make unsafe presumptions because as we have seen before, with things like XMRV, with bio-sciences presumptions can be turned on their head with one simple experiment going the opposite way from what you might presume, thus showing that reality is not what we believe it to be.

That is science and its why it helps because it reveals reality but it is also why we should be careful to understand the limits of our knowledge and try to be very logical about interpreting and designing experiments. That is why I say this shows us nothing about the reasons why PWME PBMCs accumulate phenylalanine but it does challenge us to find that out.

For full disclosure its a bit of a hobby horse of mine because I did a bioscience degree a long time ago and then I qualified as a biology teacher so I feel its my duty to point this kind of thing out but please don't hold it against me!

First of all I felt my post wasn't terribly good. I had a feeling that you were querying the ρ0 cell model. I don't know enough to say that the use of ρ0 cells is OK or not but they appear to have been using this cell line/approach for some time, so possibly that may add confidence.

I've started a blog on trying to hassle for a diagnostic test; a hobby horse of mine [https://forums.phoenixrising.me/ind...ch-theyre-working-for-you.61516/#post-1000454]. The European Union funded the development of a diagnostic test for Lyme (2 million euros).

Ron Davis is currently trying out a number of possible diagnostic tests; unfortunately not including Raman Spectroscopy. Ron reviewed these tests at the (2018) Invest in ME Conference; you can see a video here https://www.omf.ngo/2018/09/17/ron-davis-iimec13/. I asked Ben (OMF) if he'd ask Ron whether they were aware of/investigating Raman [https://forums.phoenixrising.me/ind...eds-your-help-translators.57986/#post-1000442].

I once thought cytokines were a possible diagnostic test i.e. based on the 2015 Hornig/Lipkin study but as someone pointed out you can tell there's a group effect but you cannot use if to diagnose individuals. So I have a track record of going down a route and finding that it wouldn't deliver. I still think the Hornig/Lipkin study is highly significant i.e. demonstrating inflammation but it doesn't appear to be the basis for a diagnostic test. Then there's the autoantibody stuff ---

In terms of the accumulation of phenylalanine. Ron Davis pointed out that a diagnostic test must clearly separate people with ME/CFS from people with all other diseases. At the moment they are separating people with ME/CFS from healthy controls.

Raman appears to have potential e.g. currently they've only looked at phenylalanine but they refer (from memory) to the possibility that testing for other amino acids may improve the test i.e. fewer false positives/negatives. E.g. Chris Armstrong found a bunch of other amino acids lower (and Fluge and Mella) so there are other targets. Possibly they could resolve more than one amino acid in a single analysis; thereby increasing accuracy/precision without additional expense.

I once worked as a very junior technician in a diagnostic/research/residue testing veterinary biochemistry laboratory and have a very basis qualification in chemistry from a then polytechnic. All a long time ago.

Thanks for your reply. Hopefully one of these techniques can be developed into a diagnostic test. That may help at least some of those with ME/CFS.

Regards and all the best to you

@boolybooly I've added a link to Ron Davis's talk regarding possible diagnostic tests https://www.omf.ngo/2018/09/17/ron-davis-iimec13/

 

[wigglethemouse]

Thought I should mention, the Oxford group in this study also have another project. I can't seem to find a PR thread for it so I'll put the links here. It came out a couple of weeks before the Raman spectroscopy paper and it was an ME Association update.

Research Update: Metabolomics and ME/CFS – Dr Morten and the Oxford research centre 13th August 2018
ME Association Article
https://www.meassociation.org.uk/20...-the-oxford-research-centre-13th-august-2018/
Link to PDF with more details
https://www.meassociation.org.uk/wp...Metabolomics-and-MECFS-at-Oxford-13.08.18.pdf

Of particular interest is this. I haven't seen any other researcher mention it.

Basal metabolic rate (BMR) is the rate of body energy expenditure over time. Initial comparisons indicate that basal metabolic rate is strongly correlated with two unidentified metabolites, that do not have accepted chemical names in the metabolomics database.

 

[Wishful]

It's amazing that something so important to proper body function hasn't been identified yet. Maybe we shouldn't be surprised that the researchers haven't figured out ME/CFS yet, if something that everyone has (BMR) isn't actually understood.

 

[FMMM1]

Thought I should mention, the Oxford group in this study also have another project. I can't seem to find a PR thread for it so I'll put the links here. It came out a couple of weeks before the Raman spectroscopy paper and it was an ME Association update.

Research Update: Metabolomics and ME/CFS – Dr Morten and the Oxford research centre 13th August 2018
ME Association Article
https://www.meassociation.org.uk/20...-the-oxford-research-centre-13th-august-2018/
Link to PDF with more details
https://www.meassociation.org.uk/wp...Metabolomics-and-MECFS-at-Oxford-13.08.18.pdf

Of particular interest is this. I haven't seen any other researcher mention it.

I've only looked at this briefly i.e. read above statement.

Check out Maureen Hanson's presentation at the OMF Symposium. She highlighted a somewhat surprising fact i.e. that there isn't an agreed naming system which identifies all of the metabolites. She highlighted a Canadian system which is pretty comprehensive.

In terms of the unnamed metabolites, which are a very good marker for basal metabolism, that sounds great. They will I assume know a lot about the compounds i.e. they are using a form of mass spectrometry. They must have identified the ion fingerprint (what the compounds fragment into) i.e. since they are reporting/measuring it [mass spectrometry]; they presumably have the retention time [mass spectrometry]; probably even a good idea of the molecular weight/structure etc. from the ions. These folks are world leading so they'll have the people and kit (mass spectrometry etc.) to figure out the structure etc.

If they would identify the compounds then the other researchers [e.g. Jonas Bergquist/ Hanson's group/ Japanese group (can't find it)] could check their data. I think these mass spectrometers store raw data so interrogating the raw data for a specific ion should be possible. In essence it may be possible to validate these findings in a relatively short amount of time i.e. if they share the data. OMF have an open data policy i.e. to move these things along as quickly as possible. @Wishful

You could post this on a metabolic thread (or whatever these discussions are called). I assume there's at least one e.g. for Hanson's study.

I think the Raman spectroscopy method looks good. I wonder if they could measure tryptophan using this method i.e. amino acid Phair reckons is the basis of the trap.

If you're interested in lobby for funding to progress this then this might help https://forums.phoenixrising.me/ind...ch-theyre-working-for-you.61516/#post-1001161
I've asked the European Union Committee on the Environment, Public Health and Food Safety (ENVI) if they would lobby for funding to establish whether the Raman spectroscopy method could be used as a diagnostic test for ME/CFS. This committee is currently lobbying for increased funding for research into Lyme and funding for the development of a diagnostic test for Lyme. The EU has already awarded a grant of 2 million euro/dollars for the development of a diagnostic test for Lyme. The EU has not funded any research into ME/CFS.

 

[FMMM1]

It's amazing that something so important to proper body function hasn't been identified yet. Maybe we shouldn't be surprised that the researchers haven't figured out ME/CFS yet, if something that everyone has (BMR) isn't actually understood.

If researchers would share their data then that would help speed up the process; i.e. identify the ions they have found and how.