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

http://pubs.rsc.org/en/Content/ArticleLanding/2018/AN/C8AN01437J#!divAbstract

 

From wiki:

The genetic disorder phenylketonuria (PKU) is the inability to metabolize phenylalanine because of a lack of the enzyme phenylalanine hydroxylase.

 

How good is this study?

 

And what - for dummies - does it say?

 

I understand they measured the phenylalanine content of cells and found it was elevated in ME/CFS. The method used appears to be very accurate in distinguishing between ME/CFS patients and controls.

 

Karl Morten works/specialises in Mitochondria at Oxford Uni. Is Cara Tomas the PhD student working with Julia Newton in Newcastle.

People with PKU are very severely affected. They have poor brain function and reduced life span. This condition is the reason that babies have a heel prick test a few days after birth. If they have PKU, then a low phenylalanine (an amino acid) diet in childhood can prevent/minimise abnormalities in neural development.

 

Just flicked through the paper - it seems to be a joint project between Newcastle (Tomas and Elon) and Oxford (all the others).

Also they say it is a pilot study as they only have 5 patients and 5 healthy controls. The study says that "Blood samples were collected from patients and controls following approval by the National Research Ethics Committee North East – Newcastle & North Tyneside."


Don't know anything about chemistry but this stood out:

"Other differences include bands centered at 1658 (Amide I of proteins)24 and 1440 cm-1 (CH2 and CH3 deformation vibrations of lipids)25 (Fig. 3C), both of which are higher in WT cells than in ρ0 cells (Fig. 3A). This suggests that, despite a higher accumulation of cellular phenylalanine, ρ0 cells have an overall reduced intracellular concentration of proteins and lipids due to an impaired metabolism. As phenylalanine alone might not be sufficient to characterize the pathomechanism, further biomarkers should be identified to simultaneously and more reliably identify mitochondrial dysfunction."

 

It's a really interesting study and I'm still trying to get my head around the finer details (I'm not sure my knowledge of spectroscopy from doing A-Level Chemistry a couple of decades ago quite cuts it). It's a pilot study (5 ME/CFS and 5 controls) where 4 of the 5 patients had a distinct marker of an elevated phenylalanine band. The 98% figure seems to relate to classifying individual single cell spectra taken - i.e. multiple spectrum measurements were taken for each patient. Needs replication (as always!) but very interesting.

 

I used to work with some people who had severe brain damage due to untreated PKU. They were often in a state of distress but were non-verbal so could only cry out, rock backward and forward or self-harm. It was so sad to think if they'd been screened at birth, they could have led normal lives . In adulthood, a very strict low phenylalanine diet and amino acid supplements seemed to lead to some minor improvements in their behaviours but the initial brain damage was irreversible.

 

Please explain the differences between cellular phenylalanine markers and phenylalanine RBC or urine markers?

 

98% accuracy is higher than any other results reported so far. The folks with the highest accuracy are closest to the source of the problem, right?

 

I would be cautious with the 98% figure - from what I can understand, it appears that relates to sorting between individual runs, but there were multiple runs per patient, with only 80% of the patients showing this spectral peak. That's not to pour cold water on the study - it really is an interesting and potentially very useful result - but will need replication with a far higher number of patients and controls.

ETA - missed this on my first read-through: "A larger-scale study is currently under preparation to consolidate the results from the current work."

 

Aspartame is made from aspartic acid and phenylalanine
Phenylalanine is an essential amino acid but aspartic acid isn’t

Sorry I used to analyse amino acids all day in the lab..it’s kind of imprinted in my head...I could almost remember what order they came out on the HPLC

 

Obviously it will be great if this works as an objective biomarker.

To convert phenylalanine into tyrosine you use an enzyme that is dependent on BH4. From memory we are low in BH4 or thought to be. So at first glance this does not seem a great surprise.

 

The BH4 angle might tie this into Rich von K ' s theory re folate availability. If folate availability is poor , homocysteine can form methionine via BH4 , this may compromise its availability for other reactions ?

 

Wouldn't low tyrosine also lead to low dopamine, which is implicated in central fatigue in some other illnesses?

 

I wondered the same. If phenylalanine is being diverted to other uses then that might imply difficulty in producing its normal downstream products including dopamine, noradrenaline and adrenaline - which would imply some pretty systemic symptoms like we see in ME/CFS.

Xu et al do reference other studies that indicate a reduction in 'amino acids including phenylalanine' in blood and serum:

• Armstrong et al, 2012 - found reduced glutamine and orthinine. Phenylalanine was not significantly reduced (p=0.35) on an absolute basis. However, it was correlated with the overall (reduced) level of orthinine;
  
• Armstrong et al, 2015 - phenylalanine was decreased in ME/CFS patients (p=0.003)
• Fluge et al, 2016 - the famous pyruvate paper - the only mention I can find of phenylalanine, using my highly scientific Ctrl+F approach, is a reference back to the 2015 Armstrong paper above. This paper did find lots of other reduced amino acids though
• Niblett et al 2007- lower excretion of phenylalanine in urine (p<0.003) alongside a number of other compounds. Also references an earlier study (Jones et al, 2005) that found that urinary excreta differed between CFS and depressed patients

Now, this all needs to come with a pinch of salt given the fact Xu et al is a small pilot study, but - with that caveat applied - it does seem to give additional credence to a range of studies that indicate something is up with phenylalanine in at least a subset of patients. As with all metabolic studies, whether that is a cause of ME/CFS or caused by ME/CFS (i.e. does the fact that patients move less than healthy controls cause their metabolite pattern to differ?) is still to be seen. Additionally, this finding was with PBMCs and so we don't know if any metabolic shift is replicated in other tissues, which might indicate a primarily metabolic problem, or just PBMCs, which might indicate some form of immune system activation. Still, caveats aside, it is encouraging to see findings that seem to support each other - i.e. increase phenylalanine in cells = lowered elsewhere.