Neurol Sci (2014) 35:1941–1945 DOI 10.1007/s10072-014-1867-5

ORIGINAL ARTICLE

Tryptamine levels are low in plasma of chronic migraine and chronic tension-type headache Giovanni D’Andrea • Domenico D’Amico • Gennaro Bussone • Andrea Bolner • Marco Aguggia • Maria Gabriella Saracco • Elisabetta Galloni • Valentina De Riva Antonello D’Arrigo • Davide Colavito • Alberta Leon • Francesco Perini



Received: 8 May 2014 / Accepted: 25 June 2014 / Published online: 14 July 2014 Ó Springer-Verlag Italia 2014

Abstract The primary aim of this study (TA-CH, Tryptophan Amine in Chronic Headache) was to investigate a possible role of tryptophan (TRP) metabolism in chronic migraine (CM) and chronic tension-type headache (CTTH). It is not known if TRP metabolism plays any role in CM and/or CTTH. Plasma levels of serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA), metabolite of 5-HT, and tryptamine (TRY) were tested in 73 patients with CM, 15 patients with CTTH and 37 control subjects. Of these, plasmatic TRY was significantly lower in CM (p \ 0.001) and in CTTH (p \ 0.002) patients with respect to control subjects, while 5-HIAA levels in plasma were within the same range in all groups. 5-HT was undetectable in the plasma of almost all subjects. Our results support the hypothesis that TRP metabolism is altered in CM and CTTH patients, leading to a reduction in plasma TRY. As TRY modulates the function of pain matrix serotonergic system, this may affect modulation of incoming nociceptive inputs from the trigeminal endings and posterior horns of the spinal cord. We suggest that these biochemical

G. D’Andrea  A. Bolner  A. D’Arrigo  D. Colavito (&)  A. Leon Research & Innovation (R&I) srl, Via Svizzera 16, 35127 Padua, Italy e-mail: [email protected] D. D’Amico  G. Bussone Neuroscience Department, C. Besta, Neurological Institute IRCCS Foundation, Milan, Italy M. Aguggia  M. G. Saracco Neurology Department, Asti Hospital, Asti, Italy E. Galloni  V. De Riva  F. Perini Neurology Department, Vicenza Hospital, Vicenza, Italy

abnormalities play a role in the chronicity of CM and CTTH. Keywords Chronic migraine  Tryptamine  Tryptophan metabolism

Introduction Chronic forms of primary headaches include chronic migraine (CM) and chronic tension-type headache (CTTH). Major requirements for the diagnosis of CM include the presence of headache for at least 15 days per month and a history of previous typical migraine attacks, with a portion of current attacks being classified as migraine without aura (MwwA) [1]. Similarly to CM, the IHS diagnostic criteria for CTTH include presence of headache for at least 15 days per month, classified as typical tension-type headache attacks (TTH) [1]. The pathogenesis of CM is poorly understood. The natural history raises the possibility that CM constitutes a complication and/or transformation of episodic migraine when the frequency of the attacks increases over time. It is reasonable to hypothesize that aspects of the pathogenesis of migraine without aura (MwwA) play an important role in the transformation of MwwA into CM. We have, recently, reported that an abnormality of tyrosine (T) metabolism is present in CM [2]. To date it is not known if tryptamine, the elusive amine of tryptophan metabolism, plays any role in the pathogenesis of CM and CTTH. Tryptophan (TRP) is the aminoacid precursor of indoles such as 5-hydroxytryptamine (5-HT), 5-hydroxyindolacetic acid (5-HIAA), and tryptamine (TRY). TRY together with tyramine, octopamine, and synephrine have, for a long

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Fig. 1 5-HIAA plasma levels (ng/ml) in CTTH, CM and control subjects

time, been nominated as elusive amines. The enzymes that govern the synthesis of 5-HT, 5-HIAA, and TRY are TRP hydroxylase and TRP decarboxylase respectively [3]. Numerous studies have been conducted in attempt to clarify the role of 5-HT in the pathogenesis of migraine. However the specific role of this indole still remains largely unknown [4]. TRY is a biogenic amine structurally related to 5-HT and is generated in neural and peripheral tissues in minute amounts and, for this reason, is considered the elusive amine of TRP [5]. Migraine and cluster headache have been shown to be relieved by psilocybin, a component of magic mushrooms, [5]. Whose active metabolite, psilocybin is a tryptamine analogue (N,N-dimethyltryptamine). However the specific functions of this biogenic amine was unknown until a few years ago when it was discovered that TRY is an agonist of 5-HT1A, 5-HT2A, and trace amine-associated (TAARs) receptors [6]. 5-HT1A receptor is abundant in corticolimbic regions, hippocampus, amygdala, and hypothalamus. 5-HT1A is the major somatodentritic autoreceptor on 5-HT neurons where it acts in an inhibitory fashion. 5-HT2A receptors are located in neurons of the raphe nuclei of the brain stem These neurons project widely throughout the brain regulating many functions, including that of the pain threshold [7]. TAAR receptors, on the other hand, represent a new family of G-protein coupled receptors. TAAR1 is a presynaptic receptor that modulates, in a inhibitory manner, the release of catecholamines from noradrenergic and dopaminergic fibers including those of pain matrix [8]. As a consequence, anomalies of 5-HT1A, 5-HT2A, and TAAR receptor functions have been hypothesized to play a role in the pathogenesis of migraine and tension-type headache. However, direct evidence in support of this hypothesis is

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Fig. 2 Triptamine plasma levels (ng/ml) in CTTH, CM and control subjects

still lacking. To explore this, we assessed the plasma levels of 5-HT, 5-HIAA and, TRY in plasma along with 5-HT in CM and CTTH sufferers versus control subjects (Figs. 1, 2)

Patients and methods A sample of consecutive patients presenting at Headache Centers of Neurology Departments in three Italian hospitals (Vicenza, Milan and Asti) from January 15 2011 to March 15 2011 were enrolled in this study (TA-CH, Tryptophan Amine in Chronic Headache). The inclusion criteria consisted of a history of chronic headaches lasting 1 year or more and a diagnosis of CM or CTTH. A diagnosis of CTTH was made according to ICHD-2 criteria. The diagnosis of CM included patients classified according with the Appendix Criteria of International Headache Society (IHS) [9]. Also the presence of medication overuse (MO) was based on recently revised criteria [3]. Although the diagnosis of CM using the aforementioned criteria requires the absence of MO, we first made the diagnosis of CM and later identified if a given patient also met the criteria for MO (i.e. CM ? MO), as suggested by Menack et al. [10]. Before the blood sampling, analgesic, and acute antimigraine drugs were suspended for two days in all patients, while prophylactic anti-epileptic therapy (valproate or topiramate) in the case of CM, and mainly amitriptyline in the case of CTTH, patients was maintained in a day hospital regimen, which also included hydration. Blood samples were drawn from the patients in ‘‘headache phase’’ as selected patients complained of headache attacks every day since at least 6 months. All CTTH patients were not analgesic abusers and solely in therapy with anti-

Neurol Sci (2014) 35:1941–1945

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Table 1 Mean age and sex distribution of control and patient (CTTH and CM) groups considered

Total number

Sex

Age (years)

Male

Female

Range

Mean

SD

p value

Controls

37

11 (31 %)

26 (69 %)

27-58

43.34

11.20



CTTH patients

13

2 (19 %)

11 (81 %)

19-55

38.92

12.41

0.25

CM patients

73

43 (60 %)

30 (40 %)

21-70

46.88

12.71

0.16

Table 2 Clinical characteristics of CM and CTTH patients considered CM

samples), to obtain working standard solutions at concentrations of 0, 10, and 20 ng/mL.

CTTH

Mean

SD

Range

Mean

SD

Range

History of disease (years)

11.36

10.38

1-40

14.07

2.05

1-7

Days (n°) with use of symptomatic medication per month

24.63

6.04

15-30







Total symptomatic medication (n°) per month

53.91

46.93

150







MIDAS score

52.98

19.59

25-93







Collection and sample preparation For HPLC analysis in plasma, blood was collected in vacuum sealed tubes containing ACD solution, immediately centrifuged at 3,500 rpm, for 10 min, at ?4 °C; the plasma samples were frozen at -80 °C until analysis. Both the standards and the plasma samples were processed as previously reported [4]. Apparatus and chromatographic conditions

depressive agents eventually accompanied with non frequent use of non-steroidal anti-inflammatory drugs. All patients were in a free, non-restricted, diet (Tables 1, 2). A group of 37 healthy subjects were enrolled in the control group (Table 1). The majority of the controls were selected among hospital personnel and among friends of the patients. All controls were age-matched with patients groups. There were more female in controls than in the patients group. All controls were in a free, non-restricted, diet. Subjects with headaches, diabetes, hypertension or other relevant disorders were excluded. After an informed consent was obtained from patients and controls, the plasma levels of 5-HT, 5-HIAA, and tryptamine were assessed employing HPLC methodology as previously reported [4]. HPLC analysis Chemicals and solution preparation All reagents were purchased from Farmitalia (Milan, Italy); serotonin, 5-HIAA, and tryptamine powders were purchased from Sigma-Aldrich (Milan, Italy), and cartridges for solid phase extraction (MF C18 Isolute 50 mg/1 mL, cod. 240-0005-A) from StepBio (Bologna, Italy). Stock solutions (2 mg/mL) of serotonin, 5-HIAA, and tryptamine in water were prepared and kept at -80 °C. At the time of analysis, one aliquot of stock solutions was adequately diluted in a plasma matrix (pooled from blood donor

The HPLC system consisted of two model 307 pumps (A and B) and a model 234 autosampler with a 100 ml loop, all from Gilson, Villiers-le-Bel, France. The separation was performed on a reversed phase C18 stainless-steel column (XBridge Shield 250 9 4.6 mm i.d.) packed with 5 mm particles (Waters, Duren, Germany), with a 10 9 4.6 mm i.d. Precolumn, packed with the same material. For serotonin and 5-HIAA analysis, the mobile phase flushed by pump A was an acqueous mixture of formic acid 0.1 mol/ L, citric acid 1.0 mmol/L, EDTA 0.5 mmol/L, sodium azide 500 mg/L, and diethylamine 2.5 mL/L, pH 3 (not modified); the mobile phase flushed by pump B was an almost identical solution containing 15 % acetonitrile (v/ v). Both mobile phases were flushed at 2 ml/min according to the following gradient: from 0 to 9 min pump A 100 %, from 9 to 15 min pump A 45 %, and pump B 55 %. At room temperature, the pressures were between 200 and 250 kg/cm2 and serotonin and 5-HIAA capacity factors (k0 ) were of 4.7 and 9.7 respectively. For detection, an electrochemical coulometric apparatus (Coulochem II ESA, Bedford, MA, USA) was used, Tryptamine assay was performed by HPLC with fluorescence detection. The analytical detection was performed by a fluorometer Jasco FP920 with excitation and emission lambda fixed at 285 and 340 nm, respectively. Statistical analysis Results are presented as the mean ? SD. The indolamine plasma levels were first tested for normality using the Kolmogorov–Smirnov test. All groups displayed a non-

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Table 3 Mean 5HIAA and TRY plasma levels in CM, CTTH patients versus control subjects 5HIAA (ng/ml) Mean

SD

TRY (ng/ml) p value

Mean

SD

p value –

Controls

4.22

2.03



1.83

1.08

CTTH patients

3.99

1.81

ns

0.99

0.84

0.002

CM patients

3.51

2.30

0.07

0.79

0.53

\0.001

parametric distribution; therefore the differences between groups were tested using the Mann–Whitney U test. The level of significance was set at a p value \0.05.

Results CM, CTTH, and control subjects displayed a similar age distribution, with the control group included more woman than the CM group. In all CM patients, MO was present (Tables 1, 2). Detectable levels of 5-HIAA and TRY were found in the plasma of all subjects (Table 3), while 5-HT levels were undetectable in the plasma of the majority of the subjects analysed. Table 3 reports the mean levels of 5-HIAA and TRY found in plasma of control subjects and CM or CTTH patients. The plasma levels of 5-HIAA were slightly lower, although non- significantly (p = 0.07, Mann–Witney U test), in CM patients versus controls, while those of CTTH patients were in the same range of controls and CM. In contrast, TRY plasma levels in CM and CTTH patients were significantly lower compared with those of controls (p \ 0.001, p \ 0.002, Mann–Witney U test, respectively). No statistical differences were found between the mean plasma levels of TRY in CM and CTTH sufferers. There was no significant statistical difference in the levels of 5-HIAA and TRY between male and female in all groups of subjects.

Discussion The aim of this study was to verify if anomalies in the synthesis of 5-HT, 5-HIAA, and tryptamine, all derived from tryptophan metabolism, occur in CM and CTTH patients. Results in our cohort of patients show that, in contrast to circulating 5-HT and 5-HIAA levels, those of TRY are, when compared to control subjects, significantly lower in CM and CTTH patients. Together these results raise the possibility that the tryptophan decarboxylase enzymatic activity is, unlike that of tryptophan hydroxylase, reduced in CM and CTTH sufferers.

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Tryptamine, an agonist of 5-HT1A, 5-HT2A, and TAAR receptors, is now thought to have an important role in the physiology of the CNS. The 5-HT1A receptor is a somatodendritic auto receptor that inhibits the firing, synthesis, and release of serotonin from the raphe serotonergic neurons [11]. Postsynaptic 5-HT1A receptors are, also, strongly expressed in presynaptic GABAergic interneurons of the limbic and other cortical brain regions including the orbitofrontal cortex. In these circuits the activation of 5-HT1A receptors by TRY reduces the release of GABA and activates the serotonergic orbitofrontal descending outputs to the periacqueductal gray (PAG) [12, 13]. 5-HT2A receptors, on the other hand, are densely distributed throughout the cortex, including the prefrontal cortex. In the orbitofrontal cortex, 5-HT2A mediates the excitation of serotonergic neurons connected with the PAG nuclei [14]. Together these evidences suggest that 5-HT2A, like 5-HT1A, receptors play an important role in nociception and other important functions of CNS, such as mood, anxiety, and memory. Although the pathogenesis of CM is poorly understood, one important clue is that this primary headache develops in patients with migraine without aura (MwwA) in which the number of migraine attacks increases over time. It is thus possible that the pathogenesis of CM derives from that of MwwA. In MwwA patients, there is evidence that the serotonin synthesis in the brain is increased, suggesting that the metabolism of tryptophan is altered [15]. Further support for this derives from the fact that many of the pharmacologic agents used to treat migraine interact with serotonin and its receptors [16, 17]. TRY is a agonist of 5-HT1A and 5-HT2A (much less of 5-HT1B-D) receptors. Should the low plasma levels of this amine mirror similar changes in the CNS, the here reported finding that circulating TRY levels are low in CM patients is suggestive of a dysfunction of the serotonergic system. Intriguingly, there is now evidence that 5-HT1A mediates presynaptic inhibition of GABAergic interneurons. GABA, released from these neurons, modulates the descending outputs of the serotonergic neurons from the orbitofrontal cortex inhibiting PAG [12]. One possible hypothesis is that the low levels of TRY, found in CM patients, result in an increase in GABA release and, consequently, inhibition of the functionality of PAG brainstem descending system that modulates the nociceptive transmission at the level of the spinal cord. Moreover, low levels of TRY may further increase this inhibition because of a reduced activation of 5-HT2A receptors that causes a decrease in the synthesis and release of serotonin in raphe magnum that, in turn, modulates serotonergic neurons of the orbitofrontal cortex. These abnormalities may play a role in the chronicity of the migraine.

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In CTTH, the current hypothesis is that the modulation from myofascial cranial structures at level of spinal horn, trigeminal nucleus, and supraspinal structures is impaired and that this possibly causes central sensitization and headache [18]. However, the origin of this sensitization is not clear. Some authors suggest that an up-regulation of nitric oxide synthase (NOS) together with occurrence of high and low glutamate and 5-HT levels, respectively, in platelets of these patients may support this hypothesis [19]. In contrast to this, however, we found undetectable levels of serotonin along with normal 5-HIAA levels in plasma, suggesting that the metabolic activity of tryptophan.hydroxyase is normal. Rather, in our CTTH patients, the plasma level of TRY was significantly lower than control subjects and similar to that of CM patients. As highlighted above, dysfunctional 5-HT1A and 5-HT2A receptors may hamper the inhibitory PAG control on nociceptive inputs coming from the periphery tissues to the lamina I and II of the dorsal horns and, from there, to pain-related supraspinal structures. Therefore, it cannot be excluded that the reduced TRY levels may be a reflection of the pain progression consequent to the increased frequency of the headache attacks in both CM and CTTH. Major limitations of this study include the higher prevalence of male in the CM group and the small number of patients, particularly those affected by CTTH, and controls. Another limitation is absence of a controlled diet avoiding food items containing tryptamine. In conclusion, our study shows, for the first time, that the plasma levels of tryptamine, the elusive amine of TRP metabolism, are abnormally low in CM and CTTH. Further studies are warranted to confirm this result in a large series of patients, patients with episodic migraine with high frequency of attacks, and in patients that have improved from chronic to episodic migraine (longitudinal studies).

Conflict of interest This research is partially funded by FICEF Milano Foundation. All authors declare no other disclosures.

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Tryptamine levels are low in plasma of chronic migraine and chronic tension-type headache.

The primary aim of this study (TA-CH, Tryptophan Amine in Chronic Headache) was to investigate a possible role of tryptophan (TRP) metabolism in chron...
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