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

Messages
35
Location
Western Australia
Interesting. I know glyphosate’s mechanism of action in plants is the disruption of the shikimate pathway, which is involved with the synthesis of the essential aromatic amino acids, phenylalanine, tyrosine, and tryptophan. It is purported to be safe in humans because humans do not possess the shikemate pathway. However, humans are made up of more bacterial cells than our own cells.

Early stages, but some food for thought.
 

Belbyr

Senior Member
Messages
602
Location
Memphis
https://pubs.rsc.org/en/content/articlelanding/2018/an/c8an01437j#!divAbstract

Phenylalanine can be used as a potential biomarker for diagnosis of CFS by SCRM. A machine learning classification model achieved an accuracy rate of 98% correctly assigning Raman spectra to either the CFS group or the control group. SCRM combined with machine learning algorithm therefore has the potential to become a diagnostic tool for CFS.
 

Dechi

Senior Member
Messages
1,454
I am not sure I understand this. 0 cells, what do they mean ? And what’s the date on this article ?
 

ljimbo423

Senior Member
Messages
4,705
Location
United States, New Hampshire
I am not sure I understand this. 0 cells, what do they mean ? And what’s the date on this article ?

I don't know what 0 cells are but I found this about halfway down the page-

Publication details
The article was received on 30 Jul 2018, accepted on 21 Aug 2018 and first published on 22 Aug 2018
link
 
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pattismith

Senior Member
Messages
3,932
really interesting! Thanks!

"The experimental results show that Raman bands associated with phenylalanine in 0 cells and CFS patient PBMCs were significantly higher than wild type model and healthy controls."
"As similar changes were observed in the 0 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 relate to mitochondrial/energetic dysfunction in both systems."

My understanding is that 0 cells is a model of mutant cells (which I'd like to know more about)

intracellular Phenylalanine accumulation in both cells or CFS cells could be either a cause or a consequence of respiratory chain dysfunction.

Phenylalanine accumulation is already known as a cause of dysfunction of respiratory chain:


"The results showed a reduction of SuccinateDHase and complex I + III activity in brain cortex of rats subjected to HyperPhenylAlaninemia . We also verified that Phenylalanine inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PhenylKetonUria (PKU)."

"Bioenergetics

Brain energy metabolism alterations play an important role in the pathophysiology of many inborn errors of metabolism [80-83]. In this context, energy metabolism impairment was reported in HPA animal models and patients. Significant decrease of succinate dehydrogenase (EC # 1.3.5.1) and mitochondrial respiratory chain complexes I-III activities were detected in cerebral cortex of rats subjected to experimental HPA [84], as well as decreased serum ubiquinone-10 (Coenzyme Q) concentrations in phenylketonuric patients [85]. On the other hand, Kyprianou and colleagues [86] showed no significant difference in mitochondrial respiratory chain complex I (EC # 1.6.5.3) activity in astrocytoma cells between PKU patients with or without tremor, suggesting that Phe neurotoxicity towards PKU patients does not involve the parameter of mitochondrial respiratory function. Respiratory chain complex II-III activity was also not altered in blood mononuclear cells from phenylketonuric patients [87].

Creatine kinase (CK; EC #2.7.3.2) activity, a key enzyme for maintenance of ATP homeostasis [88], is significantly inhibited in vitro by Phe and in cerebral cortex of rats subjected to experimental HPA. These results suggest another putative pathological mechanism through which Phe induces neurometabolic alterations in PKU patients [89].

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].

Phe and its metabolites also interfere on ketone bodies metabolism through inhibition of 3-hydroxybutyrate dehydrogenase (EC # 1.1.1.30) and 3-oxo-acid CoA-transferase (EC # 2.8.3.5) activities in brain of suckling rats [92]. In addition, PPA inhibited pyruvate plus malate oxidation in human and rat skeletal muscle possible due to inhibition of pyruvate dehydrogenase complex (EC # 1.2. 4.1, EC # 2.3.1.12, EC # 1.8.1.4) activity, which could collaborate to the increased lactate levels found in PKU patients [93]. Furthermore, Phe and PPA inhibited pyruvate kinase (EC # 2.7.1.40) and hexokinase (EC # 2.7.1.1) activities in adult human and fetal brain samples [94], as well as in brain of rats submitted to HPA experimental model [95,96].

Considering that metal ions (e.g. zinc, iron, and magnesium) are key components of metabolic enzymes [97], acting as regulatory entities on such proteins [15,98], and that they are deficient in PKU patients (as already mentioned), it cannot be ruled that the energy dysfunction observed in PKU may be ascribed to metal ion imbalances that disturb the activity of crucial enzymes of intermediary metabolism. Taken together, these data indicate that disturbances in cell bioenergetics homeostasis may contribute to Phe neurotoxicity observed in PKU."

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pattismith

Senior Member
Messages
3,932

ok, so this is the answer to what is a 0 cell (a cell with 0 mtDNA)
"Dr Morten and Prof Wei Huang (De artment of Engineering) from Oxford University, attempted to link mitochondrial dysfunction and ME/CFS pathogenesis by comparing the ‘fingerprint’ of a cell model containing no mitochondrial DNA (known as ‘ρ0’) to the ‘fingerprint’ of molecules from the blood cells of ME/CFS patients…"

A Newly Established Neuronal ρ-0 Cell Line Highly Susceptible to Oxidative Stress Accumulates Iron and Other Metals

RELEVANCE TO THE ORIGIN OF METAL ION DEPOSITS IN BRAINS WITH NEURODEGENERATIVE DISORDERS*



Abstract
From human neuroblastoma-derived SILA cells we have established a ρ-0 cell line that is deficient in both respiration and mitochondrial DNA. Lactate dehydrogenase activity, lactate production, and growth in the medium without glucose indicate that these cells shift from aerobic to anaerobic metabolism. Electron microscopic observations revealed abnormal mitochondria with unique cristae structures. Staining with MitoTracker dye showed that the mitochondrial transmembrane potential was reduced by 30–40% from the parent cell levels. These cells were markedly susceptible to H2O2 and died apparently by a necrotic mechanism, a process blocked by deferoxamine in the parent cells but not ρ-0 cells. Analysis by inductively coupled plasma-mass spectrometry revealed an approximately 3-fold accumulation of iron in the ρ-0 cells at confluence (n = 4–6, three clones, *p < 0.05). Iron and four other metals were all elevated in the cells of one of the ρ-0 clones and were similar to control levels in the control cybrid cells, which were replenished with normal mitochondrial DNA. Their sensitivity to H2O2 was also similar to that of the parent cells. These results indicate that a newly established neuronal related ρ-0 cell line is highly susceptible to active oxygen species and that these toxicity effects appear to be related to an accumulation of transition metals, which probably occurs through the respiratory impairment.

Iron and other transition metals exacerbate and in some cases initiate the degeneration of neurons (e.g. 1–3) through the Fenton reaction (4). In the brain of patients with Alzheimer's disease (AD),1an increase in the content of iron (5-9) and aluminum (4, 7) has been reported, and treatment of AD patients with iron chelators has been discussed (10). In the brain of patients with Parkinson's disease and Huntington's disease, iron and other metals also appear to accumulate (8, 11). It is important to note that all of these diseases show mitochondrial abnormalities to some extent (12-19), suggesting a coupling of metal accumulation with mitochondrial deficiency. More direct evidence of mitochondrial and iron association in neurodegenerative disorders comes from an increase in mitochondrial iron in the fibroblasts of patients with Friedreich's ataxia, whose responsible gene is the mitochondrial frataxin (3, 20, 21). It would also be intriguing to uncover an association of mitochondrial respiratory deficiency and cell death with an accumulation of metals because a new, pivotal, regulatory role for mitochondria in cell survival and death has emerged from a growing body of evidence (for a review, see Ref. 22).
 
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Wishful

Senior Member
Messages
5,684
Location
Alberta
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.
 

Belbyr

Senior Member
Messages
602
Location
Memphis
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.

Even though this could be a biomarker, it might not be the cause. Just like many other illnesses...
 

Lisa108

Senior Member
Messages
675
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).
 

Murph

:)
Messages
1,799
Here's what they've published. Looks like they found high levels of one amino acid, called phenylalanine. Which is a phenomenon that occurs in a known metabolic defect.

The ME Association said:
This is a new pilot study funded by the ME Association Ramsay Research Fund that introduced a relatively new technique and provided for some intriguing results.

“It is becoming clear that metabolic/energetic dysfunction plays a role in ME/CFS. More information is required to determine if these differences are driving the illness or are a consequence of having ME/CFS.

“Single Cell Raman Spectroscopy is an exciting new tool which can give a readout on aspects of intracellular metabolism. Live cells/tissue are not required, which if the approach is successful, will be a major benefit in developing a diagnostic test.”

Dr Karl Morten

About the study

Dr Morten and Prof Wei Huang (Department of Engineering) from Oxford University, attempted to link mitochondrial dysfunction and ME/CFS pathogenesis by comparing the ‘fingerprint’ of a cell model containing no mitochondrial DNA (known as ‘ρ0’) to the ‘fingerprint’ of molecules from the blood cells of ME/CFS patients.

The study involved the use of a cell imaging method called single-cell Raman micro-spectroscopy (SCRM). A light (usually from a laser) shining on a cell results in changed frequencies of photons – due to the energy exchange between the incident light and vibrations of biomolecules in cells – which are then detected and observed in the form of a Raman spectrum, named after Indian Physicist Sir C. V. Raman who earned the 1930 Nobel Prize for the discovery.

Each biomolecule has a unique ‘fingerprint’ on the Raman spectrum (shown as different length bands) and the sum of all biomolecular fingerprints in a cell can be used as a phenotype of the single cell. These fingerprints can be used to indicate changes in cellular metabolism and identify disease- related biomarkers.

This non-invasive biochemical analysis technique has an advantage over other biomarker-identifying methods as it can be performed on living cells. Also, since it is a label-free technique, the cells do not need to be radioactively labelled or stained with a dye to be imaged, so they are much closer to their natural bodily state; reflecting the intrinsic biochemical profiles of the cells with less manipulation.

However, this technique is not widely used in clinical practice at this time.

Phenylalanine – an amino acid

The researchers found that both the cells with no mitochondria and the ME/CFS patients’ blood cells had high ‘bands’ (or markers) associated with phenylalanine-like compounds, whereas the controls did not.

Phenylalanine is an amino acid (a building-block employed by the body to make important molecules) readily detectible by Raman. It is used to make many neurotransmitters, such as adrenaline (involved in the fight/flight response for example).

Although this initial exploratory study only involved 5 ME/CFS patients, it will be exciting to see where this goes when tested on a larger cohort from the ME/CFS Biobank. These findings could support a possible metabolic defect, or perhaps even a mitochondrial defect, with implications for diagnostic assessment and, ultimately, for treatment.

“As similar changes were observed in the ρ 0 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 relate to mitochondrial/energetic dysfunction in both systems.

“Interestingly, phenylalanine can be used as a potential biomarker for diagnosis of CFS by SCRM [Single-cell Raman Spectroscopy].

“A machine learning classification model achieved an accuracy rate of 98% correctly assigning Raman spectra to either the CFS group or the control group.

“SCRM combined with machine learning algorithm therefore has the potential to become a diagnostic tool for CFS.”

From: A new approach to find biomarkers in CFS/ME by single-cell Raman micro- spectroscopy (Xu, et al. 2018)

For more on the amino acid phenylalanine in ME/CFS see also for example:

  • Fluge et al. (2016) Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

  • Niblett et al. (2007) Hematologic and urinary excretion anomalies in patients with chronic fatigue syndrome

    For more information on the research being conducted by Dr Morten’s team in Oxford (funded by the ME Association Ramsay Research Fund), see:
• MEA research update: Metabolomics and ME/CFS | 13 August 2018
 
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Murph

:)
Messages
1,799
I checked Fluge and Mella's work. They found phenylalanine levels at 95% of healthy controls in serum. This rsearch is in the cell though.

Armstrong et al (2015)
found a big reduction in phenyalanine in a study that looked at blood and urine.

(That was the paper that proposed the body is using amino acids as fuel. Apparently the Phenylalanine is a clue to that. The doctor who runs the practice I go to here in Melbourne is an author on the 2015 paper, and this no doubt informed him recommending me the whey supplementation that has been so helpful to me.)
 

Mary

Moderator Resource
Messages
17,335
Location
Southern California
Mods, should these threads be merged?
You're right, they're the same study. This thread will be merged into this one which you noted, which was started last Thursday.

Thanks for catching this. I just happened across your post here. A better thing to do is to hit the report button under say the initial post and that will alert the moderators that something needs attention. And then there will be a box to state the reason for the report, and you just say something like duplicate threads, need to be merged.
 

boolybooly

Senior Member
Messages
161
Location
Northants UK
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?