Widespread Pain and altered Renal Function in MECFS Patients.

Neil R. McGregor1 PhD.

Christopher W. Armstrong2 BSc.

Donald P. Lewis3 MBBS.

Henry L. Butt4 PhD.

Paul R. Gooley2 PhD.

Correspondence:

Neil McGregorHonorary Senior Fellow Faculty of Medicine, Dentistry and Health Sciences,University of Melbourne4a Wilmot StreetMalvern East 3145Victoria, Australia.Email: neilm@unimelb.edu.au

1. Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville VIC 3010, Australia. Telephone: 61 0412469832, email: neilm@unimelb.edu.au

2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biochemistry Institute, 30 Flemington Road, Parkville, VIC 3010, Australia. Telephone: 61 383481421, email: prg@unimelb.edu.au

3. CFS Discovery, Donvale Medical Centre, 90 Mitcham Road, Donvale, VIC 3111, Australia. Telephone: 61 398414500, email: dplewis@cfsdiscovery.com.au

4. Bioscreen (Aust) Pty Ltd, 5 Little Hyde Street, Yarraville, VIC 3013, Australia. Telephone: 61 396873355, email: hbutt@bioscreenmedical.com

Abbreviations: FP Facial pain; NoFP No Facial Pain; MECFS Myalgic encephalomyelitis/Chronic Fatigue Syndrome; eGRF estimated glomelular Filtration rate; GRF Glomelular Filtration rate

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Abstract: Background: Widespread pain is noted in many patients with Chronic Fatigue Syndrome

(MECFS), Fibromyalgia (FM) and Temporomandibular disorders (TMD). These conditions usually

start as a localized condition and spread to a widespread pain condition with increasing illness

duration. Purpose: To assess the changes in biochemistry associated with pain expression and

alterations in renal function. Methods: Forty-seven MECFS patients and age/sex matched controls

had: a clinical examination, completed questionnaires, standard serum biochemistry, glucose tolerance

tests and serum and urine metabolomes in an observational study. Results: Increases in pain

distribution were associated with reductions in serum essential amino acids, urea, serum sodium and

increases in serum glucose and the 24-hour urine volume however the biochemistry was different for

each pain area. Regression modelling revealed potential acetylation and methylation defects in the

pain subjects. Conclusions: These findings confirm and extend our earlier findings. These changes

appear consistent with repeated minor inflammatory mediated alterations in kidney function resulting

in essential amino acid deprivation and inhibition of protein synthesis and genetic translation within

tissues.

Key Words: Fatigue Syndrome, Chronic; Fibromyalgia, Kidney Concentrating Ability; Facial Pain; Acetylation; Betaine;

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Introduction

Chronic fatigue syndrome (MECFS) patients frequently report widespread musculoskeletal pain or

fibromyalgia and Temporomandibular disorders (TMD) 1. Importantly patients diagnosed to have

fibromyalgia also report a high level of fatigue 2. A twin study of these conditions revealed that

fatigued twins were >20 fold more likely to be diagnosed with fibromyalgia and 4 fold more likely to

be diagnosed with TMD 3. Whilst the relationship between these conditions is observed very little is

known of their underlying etiology or pathophysiology.

A recent study of comorbidities using a group with Temporomandibular disorders (TMD) linked

TMD severity and duration with widespread pain presentation, chronic fatigue syndrome (MECFS)

and irritable bowel symptoms (IBS) 4 suggesting that they may have a common underlying

mechanism as they exacerbate at the same time. These findings are consistent with our groups earlier

studies linking the same potential comorbid conditions 5. In a previous study it was observed that

widespread pain was associated with reductions in urinary excretion of amino acids and that serum

sodium levels were associated with higher face pain score in females 6. This study also identified that

these changes were greater with the increasing duration of the illness 7. An inflammatory related

change in renal function appeared to be associated with the development of the widespread pain and

facial pain. Thus these interesting observations need to be assessed in a better designed study in an

attempt to delineate the biochemical relationships.

The objective of this paper is to assess pain severity and frequency of multiple body areas including

the face, and the related biochemistry (Blood and urine) in MECFS patients and controls. This will be

assessed as severity responses within the last 7-days and their frequency over a 12-month period.

These will be assessed for their association with inflammation and the previously observed alterations

in renal function. Subjects were assessed using standard serum biochemistry, a 24-hour urine

assessment and a blood and urine metabolome.

Methods.

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Forty-six patients with MECFS and twenty-six, fatigue free, age and sex-matched healthy individuals

were recruited. Obtaining an age and sex matched control was undertaken by placing advertisements

on University billboards and selecting from those patient of the same sex and age (±5 years) and these

patients were than assessed using the same clinical examinations. The MECFS group comprised

patients that are currently symptomatic and diagnosed as having MECFS in accordance to the

Canadian guidelines and its exclusionary criteria 8. A Depression Anxiety Stress assessment (DASS)

was used to assess their psychiatric comorbidity. Only those MECFS subjects who complied with the

criteria were included in the study. All subjects were asked to list their drugs and oral supplements.

None of the subjects were related to one another nor were they ever living together. All subjects

signed consent forms. This study was approved by the University of Melbourne human research ethics

committee (HREC# 0723086).

Clinical measures

The patients had a full clinical examination and were questioned about their illness, onset, family

histories to determine whether they complied with the Canadian ME/CFS guidelines. All subjects

completed several questionnaires, including a large symptom questionnaire developed for chronic

pain research 6. It asked patients to score how severe a symptom was in the last 7 days (0-4 scalar

response) and how frequently the symptom occurred over the last 12-month period (0-4 scalar

response), as previously published 9. The 7-day severity and 12-month frequency scores were

designed to differentiate between acute and chronic responses. These 7 day and 12 month assessments

have been assessed against the biochemistry and found to differentiate between an acute response and

a chronic event (unpublished data). These questionnaires were checked by the reception staff and the

medical clinician (DL).

Biochemistry assessments.

The sample collection and processing has previously been published 10 and are given in summary.

Subjects had a phlebotomy for serum chemistry, a 24-hour urine collection, a glucose tolerance test

and all performed a standing test during which blood pressure and transcutaneous PO 2 was measured.

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Only 9 controls had a 24-hour urine. Standard serum biochemistry was performed at a commercial

(NATA accredited) laboratory. Serum osmolality was calculated using the formula ((Sodium x 2) +

Glucose + Urea). Urine osmolality was calculated by a publish formula 11. A second urine sample

was collected upon rising by each subject at their home and stored at 4ºC and within 6 hours a blood

sample was then taken by venipuncture into BD Vacutainer® blood collection tubes. All samples

were stored at -80 ºC prior to performing an NMR analysis using a liquid-liquid extraction technique

in a Bruker Avance US 2 spectrometer. The compound libraries in the Chenomx software were used to

identify and quantitate the metabolites. Metabolite identities were confirmed using 2D TOCSY

experiments with TOPSPIN 1.3 software. Twenty-nine metabolites per blood serum sample and thirty

metabolites per urine sample were identified.

The level of creatinine in the blood is a useful guide to kidney function, and can be used to estimate

the Glomerular filtration rate (eGFR). This is a validated assessment and the criteria from the Kidney

Disease Outcomes Quality Initiative guidelines (https://www.rcpa.edu.au/Library/Practising-

Pathology/RCPA-Manual/Items/Pathology-Tests/E/eGFR) was used in this study. They are:

1. >90 mL/min/1.73m2 - normal GFR.

2. 60 - 89 mL/min/1.73m2 - mild reduction in GFR.

3. 30 - 59 mL/min/1.73m2 - moderate reduction in GFR.

Data Analysis

The metabolome data were prepared as raw data (µM) and as relatively distributed data (%) by

dividing each metabolite concentration by the total concentration of metabolites quantified in each

sample. The parametric data prior to statistical analysis was assessed for normality and log converted

if not normally distributed. All percentage data were arcsine converted for analysis. The dataset was

evaluated using Statistica for Windows Ver. 12.0 (StatSoft Inc., Tulsa, USA) using statistical

calculations, including t-tests and Pearson correlation coefficients, ANOVA and multivariate analysis.

The nonparametric data was assessed using Spearman rank correlations, Mann-Whitney U-tests or

Kruskal-Wallis ANOVA. Small sample frequency data were assessed with Fisher exact Chi square

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analysis (χ2). Multiplicity correction was carried out on the data based upon the number of variables

assessed in each statistical test.

Results.

Demographics.

In the whole group (N=72) the following pain distribution was noted. Headache 49 (C=44% v

MECFS=83%), Face Pain 22 (C=0% v MECFS=48%), Neck pain 38 (C=20% v MECFS=78%),

Shoulder 46 (C=28% v MECFS=85%), Chest Pain 31 (C=8% v MECFS=63%), Arm Pain 39 (C=4%

v MECFS=83%), Low back Pain 40 (C=16% v MECFS=78%), Leg Pain 42 (C=16% v

MECFS=83%). Eight controls (11.3% of total group) but no MECFS patients were pain free, 22

(C=60% v MECFS=13%) had 1 to 3 pain locations, 19 (C=8% v MECFS=41%) had 4 -5 pain

locations, and 22 (C=0 v MECFS=28%) had pain in ≥6 locations. Face pain was only found in the

MECFS patients in this sample so to examine the pain biochemistry we chose to divide the MECFS

group on this basis for the analysis as it had less potential confounders and face pain had a relatively

unique pain associated biochemistry. The other analyses are available as supplementary data.

Table 1 shows the patient demographics for the MECFS patients divided on the basis of presence or

absence of face pain (FP and NoFP) and the control group. No differences were found for sudden v

gradual onset or triggers at onset, such as infections or trauma.

Symptoms.

Table 1 also shows the distribution of body pain symptoms across the groups. Whilst the widespread

pain distribution and the cumulative severity scores were increased in the FP group compared with

both the NoFP and control groups this only related to increases in only five areas of pain (headaches,

face, neck/shoulder and low back). Table 2 shows the distribution of potential inflammatory or

exacerbating symptoms for each group. Table 3 shows the correlation analysis of the various

potentially inflammatory or exacerbating symptoms associated with face pain scores, body

distribution scores and the cumulative pain scores within the MECFS patients. The 7-Day febrile

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score was associated with pain symptom expression but not the 12-month frequency score. Food

intolerance, including cases of gluten intolerance, were associated with widespread pain. Interestingly

irritable bowel symptom scores did not correlate with widespread pain within the MECFS patients

even though it did if the controls were included in the correlation analysis.

Assessment of renal function (Estimated Glomerular Filtration Rate - eGFR).

Only one NoFP patient had an eGFR of 59 and 31 of the 43 MECFS patients (72.1%) had an eGFR

between 60 and 89 suggestive of mild changes in renal function (74% abnormal). Table 4 shows the

eGFR for each group. Table 5 shows the eGFR was positively correlated with the pain scores,

showing that a reduction in renal function was not related to the pain scores. No association was

found between the Body Mass index (BMI), the drugs being taken and the eGFR.

Biochemistry.

Table 4 shows a summary of the renal function measure differences between the groups. A pattern of

increased glucose and 24-hour urine volume, and reduced urea, lysine and, phenylalanine were

observed to relate to increased pain scores. None of these factors correlated with the subjects BMI.

Three subjects (all FP) had a serum urea below the normal two standard deviation range for

Caucasians in the US (Males 3.46-11.86 mmol/L; females 3.21-10.33 mmol/L) 12. Multiple regression

modelling was used to identify the metabolites of significance for pain development and they were

predominately a fall in acetate and an increase in betaine (supplemental data available). However, the

increasing pain body distribution was also associated with increases in glucose and falls in acetate,

lysine and phenylalanine.

Table 5 (a heat map) shows the correlation analysis of the metabolites against the pain scores. For the

whole study group, the 7-Day severity scores strongly negatively correlated with serum

phenylalanine, acetate and osmolality, and urine urea and acetate. For the 12-month frequency scores

serum phenylalanine and urine urea were the strongest correlates followed by both serum and urine

acetate (all negative). For the MECFS patients no metabolite reached statistical significance at p<.01.

The 24-hour urine volume correlated positively with 7-day face pain severity.

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Table 6 (a heat map) shows the changes in the metabolite associations within the 3 study groups and

the assessment of their statistical differences between the groups. The patterns of metabolite

correlations were significantly different between the groups. Discriminant Function analysis models

using the variables within table 5 revealed significant models for:

FP v Control (Wilk’s Lambda= .353, F(11,35) =5.83 p<.0000) with 3 variables identified:

Betaine (p<.001), Phenylalanine (p<.03) and Glucose (p<.04).

NoFP v Control (Wilk’s Lambda= .611, F(11,38) =2.19 p<.04) with 1 variable identified:

Glucose (p<.04).

NoFP v FP (Wilk’s Lambda= .524, F(11,35) =2.88 p<.008) with 2 variables identified: 24-

hour urine volume (p<.008) and Betaine (p<.02).

These analyses identify glucose, phenylalanine, betaine and 24-hour urine volume as the best

predictors of the group differences.

Discussion.

This paper has confirmed many of the associations identified in other studies, namely the associations

with: face pain and widespread pain; MECFS; irritable bowel syndrome; and inflammatory/infective

events (the subjective feelings of infection) 5. Assessment of the findings within the MECFS cohort

did not confirm the association between irritable bowel and face or widespread pain. However, it did

identify a potential association with food intolerance, possibly gluten intolerance, which some may

report as irritable bowel.

This paper has confirmed our earlier finding of a change in renal function, urea and amino acids being

associated with face and widespread pain 6;7. The fall in urea was associated with a reduction in serum

essential amino acids, in particular phenylalanine and lysine, and the non-essential amino acids, which

facilitate urea production within the urea cycle. It was not associated with changes in the BMI or

eGFR. These data are consistent with the observations of urea cycle associated amino acid differences

in fibromyalgia and MECFS 6;10;13;14. In addition, the fall in urinary urea was associated with an

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increased excretion of total metabolites in the urine and an increase in urine volume suggesting an

alteration in urea related kidney concentrating activities. This was also previously observed 7.

These changes are very similar to the renal changes seen in Central Diabetes Insipidus 15, protein-

calorie restriction 16 and infection/inflammatory mediated events 17-19. However, no subjects had

Diabetes Insipidus or were protein calorie restricted, and all had average BMI’s, which supports an

inflammatory origin. The FP and widespread pain group had a higher level of reporting of infectious

events and scalar responses (table 2) as previously reported 5. The renal changes are consistent with

those reported for kidney function in sepsis 17;20, which appear to involve inhibition of aquaporin and

urea transporters in the kidney leading to polyuria and metabolite loss 19. In support of this, urea is

produced in the kidney by arginase 2 and deprivation of protein will induce a fall in urea and arginase

activity 21. The Arginase II gene (ARG2) has NF-Kappa-β binding sites in the promoter region and is

altered by inhibition of NF-Kappa-β 22. There is evidence for an NF-Kappa-β/ inflammation mediated

alteration in activity 23 and its levels have been reported to be elevated in MECFS patients 24. In that

study there were significant positive correlations between the production of NF-Kappa-β and the

severity of illness as measured with the FibroFatigue scale and with symptoms, such as aches and

pain, muscular tension, fatigue, irritability, sadness, and the subjective feeling of infection 24. The

action of upregulation of kidney arginase during inflammation is to remove excess nitrogen from the

system after its release from muscle and tissue stores. Based upon these observations we originally

proposed a model of repeated inflammatory stimuli initiating an aminoaciduria and electrolyte loss

which resulted in the development of an amino acid deficiency and a change in electrolyte balance

and the subsequent development of widespread pain 6. This study supports that hypothesis.

A fall in the essential amino acids, phenylalanine and lysine, would interfere with intracellular

protein synthesis and DNA translation through a Eukaryotic initiation factor kinase 2 (EIF2)

mechanism 25. Possibly also involved would be insulin resistance mediated amino acid uptake as

evidenced by the association with increasing blood glucose 26;27. The MECFS patients do have an

anomaly in insulin excretion in this study (Data to be published separately) which supports this

conclusion.

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Also involved in the wide pain development is the fall in acetate, which indicates a disturbance of

acetylation, and the increase in betaine, which may indicate a disturbance in methylation 28;29. The

reduction in lysine observed, only in the pain subjects, may be of critical importance, as acetate

binding to lysine residues within enzymatic proteins regulates many enzyme activities 30 including

inflammation 31. The increase in betaine has been observed in an animal model of allergic encephalitis

along with a fall in acetate 32. Betaine may be important in the development of the widespread pain

through a central sensitization mechanism as it inhibits gamma-amino butyric acid (GABA) transport

33. This may be the basis of the observed alterations in spinal GABA and its receptors with central

sensitization 34-36 and the claimed therapeutic benefits of GABA reuptake inhibitors in the treatment of

pain 37;38. Studies are required to confirm these interesting findings.

In our original study we found that the serum sodium level was inversely correlated with the face pain

scores in females but not correlated with widespread pain 6. In this study we found the same

relationship between serum sodium and face pain scores and also no relationship with widespread

pain. A fall in arginase II expression in the kidney has been linked to variation in salt tolerance in

Dahl salt sensitive rats 39 and alteration in sodium channel Nav1.6 has been linked to the development

of neuropathic pain in diabetes 40. In this study a fall in serum osmolality, a measure of electrolyte

balance, was a major predictor of 7-day pain responses, but it was not as strong for the 12-month

frequency scores. Two important studies have associated urea, sodium and/or betaine with regulation

of water channel (aquaporin 1-3) expression within the kidney 41;42 suggesting the relationship

between urine volume, sodium and betaine observed in this study is of importance. In further support

of this, changes in expression of aquaporin 1 have been associated with central sensitization within

the spinal column 42. Changes in Betaine may also be associated with polymorphic variation in the

methylation/one carbon pool enzymes 43. How these changes in sodium, urea and aquaporin may

relate to pain is not known but aquaporins are expressed within various tissues including the

temporomandibular joint and vary with arthritis 44.

This study was designed to investigate metabolic changes in MECFS subjects using a discovery

hypothesis and not a specific hypothesis driven method to assess specific biochemical events. This

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study with these limitations has resulted in the development of a hypothesis which now requires to be

assessed by a typical hypothesis driven process. Whilst the study size is small it reproduced the earlier

findings but should be reproduced with a larger sample to reconfirm the findings. The use of self-

reported symptoms may introduce a recall bias within the patient and in a larger study each of the

variables found to be associated with the pain severity and distribution need to be evaluated by other

means. Studies investigating the genetic polymorphic variation and their association with the

biochemical events should allow the development of the understanding of the mechanisms of pain

development and the development of appropriate therapies based upon the underlying biochemistry.

Conclusions.

This study has confirmed and extended the findings of our previous studies showing an

association between altered amino acid excretion, urine volume, serum sodium, osmolality and

increases in facial and widespread pain expression in MECFS patients. The falls in serum essential

amino acids appeared involved in the development of the changes in urea, urine volume and sodium.

The changes noted appear consistent with alterations in kidney function induced by an inflammatory

process and/or sepsis. Widespread pain appears to be a consequence of the repeated inflammatory

driven changes in renal concentrating activity leading to essential amino acid depletion. Well-

designed studies evaluating these important factors are warranted.

Acknowledgements: The authors of this work would like to thank the nursing and administrative

staff at the CFS Discovery clinic for their important help throughout this study. The work was

supported by grants from the Judith Jane Mason & Harold Stannett Williams memorial foundation

(The Mason Foundation) and equipment grants from the Rowen White Foundation and the State of

Victoria.

Conflicts of Interest: There are no conflicts of interest.

Compliance with ethics: The study was approved by the University of Melbourne human research

ethics committee (HREC# 0723086).

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