Altered amino acid homeostasis in subjects affected by fibromyalgia
Laura Bazzichi a,⁎, Lionella Palego b, Gino Giannaccini b, Alessandra Rossi b, Francesca De Feo a,Camillo Giacomelli b, Laura Betti b, Laura Giusti b, Giovanni Mascia b,
Stefano Bombardieri a, Antonio Lucacchini b
a Department of Internal Medicine, Division of Rheumatology, University of Pisa, Via Roma 67, 56126 Pisa, Italyb Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
Received 7 January 2009; received in revised form 27 February 2009; accepted 28 February 2009Available online 10 March 2009
Abstract
Objectives: To evaluate plasma amino acid (AA) concentrations in patients affected by fibromyalgia (FM) and to study the relationshipsbetween their levels and FM clinical parameters.
Design and methods: 20 AAs were assessed in 34 FM patients and in 18 healthy volunteers by means of a modified version of the Waterspicotag method.
Results: Significant lower plasma taurine, alanine, tyrosine (Tyr), valine, methionine, phenylalanine and threonine concentrations, and the sumof essential AAs were observed in FM patients vs healthy controls (Pb0.05). Tyr CAA' ratio and the sum of AAs competing with tryptophan forbrain uptake were significantly reduced in FM (Pb0.05). A significant correlation was found between FM clinical parameters and certain AAs.
Fibromyalgia (FM), as defined in the 1990 American Collegeof Rheumatology (ACR) criteria [1] is a chronic, generalizedpain condition with characteristic tender points on physicalexamination, often accompanied by a number of associatedsymptoms such as fatigue, sleep disturbance, headache, irritablebowel syndrome and mood disorders.
Pathophysiological hypotheses of FM include impairment inthe functioning of the hypothalamic–pituitary axis and alte-rations in neuromodulators and neurotransmitters such as sub-stance P (SP), nerve growth factor (NGF), N-methyl-D-aspartate, norepinephrine (NE) and serotonin (5-HT) [2–4].Substance P and nerve growth factor resulted in an increase inthe cerebrospinal fluid (CSF) of patients with primary fibro-myalgia but not in fibromyalgia patients with associated
painful inflammatory conditions (secondary fibromyalgia)[5–7]. Substance P is a putative modulator of nociception andNGF is the neurotrophic factor that regulates SP synthesis inprimary afferent C-fibres, structure thought to transmit painstimuli. 5-HT and tryptophan concentrations were found to bedecreased in serum and CSF of patients with FM [8]. 5-HT istheorized to have a function in stage 4 sleep and pain thres-hold [9], besides its implication in psychiatric disorders suchas depression, anxiety, and obsessive compulsive disorder isoften present in fibromyalgic patients.
Considering that tryptophan is the amino acid (AA) pre-cursor of serotonin synthesis and that plasma tryptophan mayreflect the status of tryptophan and serotonin in the brain,several authors measured free plasma tryptophan in patientswith FM. Moldofsky and Warsh [10] found that free plasmatryptophan is inversely related to morning pain in 8 fibromyal-gic patients. Russell et al. [11] reported that the concentrationsof serum tryptophan and 9 other amino acids (alanine, histidine,lysine, proline, threonine, serine, taurine and phosphoserine)
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were significantly lower among 20 patients with fibrositis/fibromyalgia syndrome, compared to 20 matched controls.Yunus et al. [12] measuring plasma tryptophan and its transportratio, together with other twenty-one amino acids, in patientswith fibromyalgia found that the transport ratio of tryptophanwas significantly decreased in FM patients compared to thecontrol group and the plasma tryptophan level was lower inFM patients than in healthy controls showing a trend towardssignificance. Also plasma histidine and serine levels werefound to be significantly lower in patients with FM than incontrols.
Yunus et al. [12] suggested that because tryptophan crossesthe blood/brain barrier via a transport mechanism that is sharedwith other branched chain large neutral amino acids, thetransport ratio of these amino acids provides a more meaningfulindex of their entry into the brain than the plasma concentrationsof any one of them alone.
Some authors suggested that a malfunction of energymetabolism may be present in some of the muscle fibres offibromyalgia patients [13,14]. It is hypothesized that muscleenergy depletion could by itself evoke many of the symptoms offibromyalgia [13,15]. A factor that could contribute to muscleenergy depletion is reduced plasma concentrations of thebranched chain amino acids (BCAAs), leucine, valine andisoleucine [14]. There is evidence that BCAA supplementationdecreases muscle catabolism and has ergogenic values, theirinfusion displays several CNS-mediated effects including anti-nociceptive action in healthy subjects but not in FM patients [16].
Recent advances support the involvement of peripheral andcentral sensitization disturbances of pain-related processesinvolving the increased transmission of the excitatory aminoacid glutamate. Few studies support the implication of this aminoacid in chronic migraine and primary fibromyalgia and demon-strated increased levels of glutamate in the cerebrospinal fluid ofaffected patients [17]. Larson et al. [8] did not show any variationin the levels of excitatory amino acids (EEAs) in the CSF of FMpatients compared to controls, while they found significantcorrelations between the concentrations of some EEAs (arginine,taurine and glycine) and the tender point index (TPi).
A heterogeneous picture exists in literature about AA levelsin FM patients, in light of these results we decided to evaluateplasma AA levels in FM patients and to study the relationshipsbetween their levels and age, FM clinical and diagnosticparameters. Because amino acid plasma levels have been foundaltered in psychiatric disorders [18,19], the presence ofpsychiatric comorbidity in fibromyalgic patients might repre-sent a confounding factor during the elaboration of results. Forthis reason we have recruited a population of FM patients with anegative history of psychiatric disorders.
Methods
Subjects
34 patients affected by fibromyalgia (29 F, 5 M), aged49.56±13.82 years (mean age±S.D.) were enrolled. Patientswere recruited and clinically classified at the Division of
Rheumatology, University of Pisa (St. Chiara Hospital)according to the 1990 American College of Rheumatologycriteria (ACR criteria) [1], which include: pain for more than3 months from all of the four body quadrants, axial skeletalpain and pain upon digital palpation of at least 11 out of 18specific bilateral points. Healthy volunteers (17 F, 1 M, 39.35±12.76 years) were recruited from the Transfusion Centre of theSt. Chiara Hospital (Pisa) and they were all routinelymonitored blood donors. Exclusionary criteria for normalvolunteers were: any of the above ACR criteria for fibro-myalgia; use of any medication. Exclusionary criterion forpatients was: the presence of a major clinical condition otherthan fibromyalgia. The patients and controls with recent or pasthistory of psychiatric disorders and pregnant females wereexcluded from the study. All patients maintained their usualdiet or physical activity and they had a drug wash out period ofat least 2 weeks before blood sampling.
Written consent was obtained from all subjects after a fullexplanation of the study.
Evaluation of clinical parameters
Tenderness at tender points was evaluated in each subjectusing the Fischer dolorimeter [20]. A rheumatologist appliedthe instrument at a rate of 1 kg/s and the patient was ins-tructed to say when this procedure became painful. The painthreshold was calculated for 18 points, and the tender point(TP) count was determined by the number of tender pointsthat had a threshold of ≤4 kg/cm2. The total fibromyalgictender point score (right+ left) was used in the statisticalanalysis.
To estimate the impact of fibromyalgia on the quality of life,all the patients received a “Fibromyalgia Impact Question-naire” consisting of 10 items. The resulting score (FIQ totalscore), which indicates the impact of the disease on life,ranged from 0 (no impact) to 100 (maximum impact). Foreach patient an evaluation was also made of fatigue by meansof a visual analogic scale (VAS, 0–10). Each patient wasasked if they had frequently suffered from unrestful sleep(frequent and/or early awakening as well as inability to fallasleep) [21]. Also the duration of disease (years) was takeninto consideration for FM patients.
Blood collection, plasma separation and deproteinization,plasma amino acid identification
Blood sodium–EDTA treated samples were collected andimmediately centrifuged at 2600 ×g for 15 min. Plasma aliquotswere diluted 1:1 with HCl 0.1 N, containing 100 μM internalstandard (Iss: beta-alanine, alpha-aminobutyric acid, norleu-cine). After mixing this plasma dilution, acetonitrile (2.8 vol.)was immediately added to precipitate plasma proteins. Sampleswere maintained on ice for 15–20 min and centrifuged for15 min at 12,000 ×g, 10 °C. The amino acids were determinedby means of a modified version of the Waters picotag method(Waters S.p.a.) using phenylisotiocianate as the derivatizingagent (our unpublished data).
Transport ratio for tryptophan was calculated as follows[22,12]: tryptophan transport ratio=micromolar concentrationof plasma tryptophan / sum of micromolar concentrations ofother plasma large neutral amino acids. The same formula wasused for the determination of the transport ratio of tyrosine.
For the statistical analysis of the data, Mann–Whitney testand Spearman correlation were used, setting the significancelevel at Pb0.05.
Results
The demographic data and clinical characteristics of the FMpatients are shown in Table 1.
Tables 2a–d show the results of amino acid measurements:patients with fibromyalgia had significantly lower plasmataurine, alanine, tyrosine, valine, methionine, phenylalanine andthreonine concentrations than healthy controls. Histidine levelswere also lower without reaching the statistical significance(P=0.06) (Table 2a). Moreover, the sum of essential AAs wassignificantly lower in fibromyalgia than in normal controls.
Table 2aPlasma concentrations of essential plasma amino acids (AAs), sum of essentialAAs and branched AAs (BCAAs) in patients with fibromyalgia and normalcontrols.
BCAAs: the sum of branched amino acids valine, leucine and isoleucine.(⁎) Statistically significant differences; (°) differences near statisticalsignificance.Results are shown as mean±S.D. Amino acid levels are expressed in μmol/L(μM).
There was a trend toward significantly lowered plasma BCAA'concentration in fibromyalgia patients (P=0.05). The trypto-phan CAA' ratio was not different between patients andcontrols, instead the Tyr CAA' ratio and the sum of AAscompeting with Trp for brain uptake (CAA Trp) weresignificantly reduced in FM (Table 2c).
In FM patients, age correlated positively with tryptophan(r=0.48, P=0.023), glutamine (r=0.367, P=0.036) and taurine(r=0.342, P=0.05), while with alanine (P=0.08), lysine(P=0.07) and glutamic acid (P=0.079) the results are nearsignificance.
A significant and positive correlation was found betweenglutamate and the number of TPs in FM patients (r=0.495,P=0.0139). FIQ correlated negatively with plasma histidine(r=−0.415, P = 0.020) and phenylalanine (r=−0.375,P=0.038), while a slight tendency toward significance wasfound for isoleucine (r=−0.349, P=0.086).
Patients have been separated according to the presence/absence of unrestful sleep. Patients with unrestful sleep (N=22)showed significantly elevated plasma concentrations of serine(86.86 vs 76.98, P=0.044 Mann–Whitney test), and taurine(35.07 vs 28.68, P=0.039), while glutamine (462 vs 409.4,
Table 2cPlasma concentrations of competing amino acids (with Trp and Tyr) andtransport ratio for Trp and Tyr.
FM patients Normal controls P
CAATrp (⁎) 517.3±88.05 589.6±130.5 0.0488CAATyr 536.3±104.9 584.5±110.9 N.S.Trp CAA ratio 0.16±0.07 0.14±0.04 N.S.Tyr CAA ratio (⁎) 0.12±0.02 0.14±0.03 0.0329
(⁎) Statistically significant differences.Results are shown as mean±S.D. Amino acid levels are expressed in μmol/L(μM).Trp: tryptophan, Tyr: tyrosine. CAATrp: sum AA competing with Trp for brainuptake (Tyr, Phe, BCAAs). CAA Tyr: sum AA competing with Tyr for brainuptake (Trp, Phe, BCAAs). Trp CAA ratio: the tryptophan CAA ratio. Tyr CAAratio: the tyrosine CAA ratio.
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P=0.093) and arginine (86.91 vs 72.28, –>P=0.05) showed atrend to higher plasma levels (Table 3).
No-significant correlations were found between plasmaamino acids and VAS fatigue or disease duration. No-significantcorrelations were found between age and AA levels in normalcontrols.
Discussion
FM syndrome is characterized by an abnormal sensory pro-cessing of pain signals and is thought to arise from a combi-nation of interactions between neurotransmitters, externalstressors, behavioural constructs, hormones, and the sympa-thetic nervous system. The measurement of serum/plasmaamino acid concentrations has been helpful in understanding thepathogenesis of several clinical disorders characterized by lowlevels of selected amino acids in the blood due to inadequategastrointestinal absorption or to faulty conservation of thekidney or to a combination of both defects [23–25]. We studiedthe plasma amino acid concentrations to investigate the aminoacid homeostasis in fibromyalgic patients.
Our results showed significant lowered concentrations ofplasma taurine, alanine, tyrosine, valine, methionine, phenyla-lanine and threonine in FM patients with respect to normalcontrols. Also concentrations of histidine were lower in FMpatients with a trend toward significance.
In literature, a general trend of lowered plasma or serumconcentrations in FM patients with respect to healthy controlshas been shown. Yunus et al. [12] found significant loweredconcentrations of the following plasma amino acids: serine andhistidine and a trend to lower concentrations of arginine,methionine, threonine and tryptophan. Russell et al. [11] foundsignificant lower serum concentrations of: alanine, histidine,lysine, proline, serine, taurine, threonine and tryptophan. Maeset al. [14] found lower concentrations of the following plasmaamino acids: phenylalanine, valine, leucine, isoleucine and atrend for tryptophan.
We showed that the sum of essential AAs was significantlylower in FM patients than in normal controls, and also plasmamean concentrations of BCAAs were lower in patients. AlsoMaes et al. [14] found significantly lower plasma concentrationsof BCAAs, which may be supportive of the muscle energydepletion hypothesis of fibromyalgia. Moreover there are manystudies which showed that BCAA supplementation maydecrease muscle catabolism and has ergogenic values [26,27].In fact provision of BCAAs may also decrease central fatigue,through increased competition for the cerebral uptake mechan-ism of tryptophan [28]. We have also supported the muscle
energy depletion hypothesis in a previous research [29] showingsignificant lower ATP levels and higher calcium and mag-nesium concentrations inside platelets of FM patients.
The tryptophan (Trp) CAA' ratio is an indicator for theavailability of Trp to the brain and hence for the 5-HT synthesisin the brain; we did not find any differences of plasma Trpconcentration or Trp ratio, according to Maes et al. [14] but incontrast with Yunus et al. [12] and Russell et al. [11]. Instead,we found for the first time in FM patients significant loweredconcentrations of: plasma tyrosine, the sum of AA competingwith Trp for brain uptake, and Tyr CAA ratio.
Because tyrosine is the precursor of the catecholaminenorepinephrine, epinephrine and dopamine these evidences letus suppose an impairment at the level of catecholamine synthesisin FM patients, differently from those results which underline animpairment in the serotonin synthesis. In accordance with thishypothesis, recent studies support the hypothesis of a dysfunc-tion of dopaminergic transmission in FM patients [30] indicatingthat it represents a relevant target for the treatment of fibro-myalgia. Dopaminergic agonists of receptors D3/2 have beenefficacious in decreasing the symptoms of fibromyalgia. Hol-man et al. [31,32] in a pilot double blinded study, reported adecrease of pain evaluated by visual analogic scale (VAS), andimprovement in other parameters such as rigidity and myalgicscore of TPs after the treatment with ropinirole and pramipexole.Recently, Buskila et al. [33] have reported on an associationbetween fibromyalgia and the D4 dopamine receptor exon IIIrepeat polymorphism and a relationship to novelty seekingpersonality traits. Interestingly, it was shown [34] that dopami-nergic rather than serotonergic neurotransmission is altered infibromyalgia, suggesting increased sensitivity or density of D2
dopamine receptor in fibromyalgia patients. Also the beneficialeffects of the use of new and selective norepinephrine and
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serotonin reuptake inhibitors (duloxetine, milnacipram anddesvenlafaxine) in FM patients [35–37] support the hypothesisof a dysfunction of catecholaminergic system in this pathology.
The lower concentrations of tyrosine may also influence thesynthesis of the thyroid hormones triiodothyronine (T3) andthyroxine (T4) which are derived from tyrosine in the colloid ofthe thyroid. Aside from being a proteogenic amino acid,tyrosine has a special role by virtue of the phenol functionality;it occurs in proteins that are part of signal transductionprocesses functioning as a receiver of phosphate groups thatare transferred by way of protein kinases (so-called receptortyrosine kinases) and phosphorylation of the hydroxyl groupchanges the activity of the target protein. Moreover manystudies indicated the use of tyrosine in particular conditionssuch as stress [38], fatigue [39] and insomnia [40,41].
In accordance with Maes et al. [14] we also observed sig-nificantly lower plasma levels of the tyrosine precursor phenyl-alanine in FM patients. Besides its role in catecholaminesynthesis, phenylalanine concentrations may affect hepaticprotidogenesis by retarding protein degradation in the liver [42].
Amongst the essential amino acids, we observed low levelsof threonine in FM patients. Since threonine is a potentialsource of glycine and serine, this could indicate an impairedreserve of the synthesis of these two amino acids. Further it hasbeen showed that chronic low levels of threonine can lead tohypoglycemic symptoms of fatigue, headaches and anxiety[43].
Particular interest should be given to the here-reporteddecrease in the sulfur-containing amino acids taurine andmethionine, linked to both energy metabolism and neurotrans-mission. Methionine is one of the main sources of sulfur in thebody and is the amino acid from which sulfur compounds ofinterest in humans are synthesized. It cannot be synthesized byanimals but generally non-restrictive diets supply adequateamounts. Methionine prevents fatty liver through its ability totransmethylate to form choline, necessary to prevent fatty liverdisease and eventual cirrhosis [44]; lowers circulating levels ofacethaldeide after ethanol ingestion [45]; promotes detoxifica-tion of xenobiotics via the sulfation pathway. Low levels ofmethionine have been found in patients affected by AIDS andthere are reports of its effectiveness in the treatment ofParkinson's disease [46] and acute pancreatitis [47].
Nevertheless the sulfur compounds s-adenosylmethionine(SAMe), dimethylsulfoxide (DMSO), taurine and reducedglutathione have clinical application in the treatment of anumber of conditions such as depression [48], arthritis,interstitial cystitis, athletic injuries, congestive heart failure,diabetes, cancer AIDS and fibromyalgia [49,50].
Taurine is a conditionally essential sulfonated beta aminoacid derived from methionine and cysteine metabolism. It ispresent in high concentrations in most tissues, particularly inproinflammatory cells such as polymorphonuclear phagocytesand in the retina [51]. Retinal pathologies have been reportedfor animals and human deficient in taurine [52]. With theexception of cow's milk, taurine is widely distributed in foodsfrom many animal (but not plant) sources [53]. Metabolicactions of taurine include bile acid conjugation, detoxification,
membrane stabilization, osmoregulation, and modulation ofcellular calcium levels [54,55]. Clinically, taurine has been usedwith varying degrees of success in the treatment of thefollowing conditions: cardiovascular diseases, hypercholester-olemia, epilepsy, Alzheimer's disease, hepatic disorders,alcoholism and cystic fibrosis [53,54].
Moreover, interestingly, sulfur-containing amino acids areprecursors of either catabolic or anabolic pathways ofcathecolamines and these pathways can integrate with thepurine nucleotide signalling [29].
Amongst non essential amino acids, we observed low levelsof alanine in FM patients. This amino acid plays a key role inthe glucose–alanine cycle between tissues and liver, carryingamino acid nitrogen from muscle to liver where its carbonskeleton is converted to glucose via gluconeogenesis. Thispathway seems therefore altered in FM patients.
We found an interesting correlation between clinical indexand some amino acids: glutamic acid correlated significantlywith TPs, histidine and phenylalanine correlated negativelywith FIQ. Moreover patients with unrestful sleep showedelevated plasma concentrations of serine, taurine arginine andelevated but not significant concentrations of glutamine.
The findings of significant relations between clinical indexand certain amino acids may support the hypothesis of ametabolic disturbance in FM. Fibromyalgic patients might haveinadequate gastrointestinal absorption or a faulty conservationof the kidney or a combination of both defects. Some authors[13] have suggested that the lowered serotonergic metabolismin FM is related to a defective absorption of the precursor aminoacid tryptophan from the gut. Nevertheless irritable bowelsyndrome is a documented clinical characteristic presented inhigh percentage in patients with fibromyalgia [4]. All theseevidences support the hypothesis of an altered amino acidhomeostasis in subjects affected by FM.
In summary the major findings of this article are: patientswith FM have significantly lower plasma concentrations of thesum of essential amino acids, in particular lower concentrationsof the sulfur-containing amino acids; lower concentrations ofthe sum of BCAAs, and lower tyrosine and Tyr CAA ratio thannormal controls.
We did not find any significant differences in plasma Trp andthe Trp CAA ratio, thus, our results are not in agreement withthe hypothesis that a deficiency in serotonergic neuronalfunctioning may be related to the pathophysiology offibromyalgia. Literature is controversial at this regard probablythis is attributable to a different recruitment of patients, in somecases findings in FM were found when all individuals with thisdisorder were studied, but not when individuals free ofpsychiatric comorbidities were studied, suggesting that someof the findings may track more closely with psychiatriccomorbidity than inherent features of FM.
Our results indicate a possible impairment of cathecolami-nergic system and suggest probable defects of gut malabsorp-tion of certain important groups of amino acids: essentially, theergogenic BCAA and the sulfur-containing amino acids in thefibromyalgic syndrome. These results are representative of agroup of FM patients without recent or past history of psy-
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chiatric disorders, thus, further studies are needed to investi-gate the amino acid homeostasis in FM patients with psy-chiatric comorbidities.
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