widespread pain and renal function web viewsmall sample frequency data were assessed with fisher...
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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: [email protected]
1. Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville VIC 3010, Australia. Telephone: 61 0412469832, email: [email protected]
2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biochemistry Institute, 30 Flemington Road, Parkville, VIC 3010, Australia. Telephone: 61 383481421, email: [email protected]
3. CFS Discovery, Donvale Medical Centre, 90 Mitcham Road, Donvale, VIC 3111, Australia. Telephone: 61 398414500, email: [email protected]
4. Bioscreen (Aust) Pty Ltd, 5 Little Hyde Street, Yarraville, VIC 3013, Australia. Telephone: 61 396873355, email: [email protected]
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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