Serum level of semicarbazide-sensitive amine oxidase in children with ADHD

1744-9081-2-5 1744-9081 Short paper Serum level of semicarbazide-sensitive amine oxidase in children with ADHD Roessner Veit vroessn@gwdg.de Uebel Henrik huebel@gwdg.de Becker Andreas abecker4@gwdg.de Beck Georg georg.beck@klab.imed.uni-erlangen.de Bleich Stefan stefan.bleich@psych.imed.uni-erlangen.de Rothenberger Aribert arothen@gwdg.de

Department of Child and Adolescent Psychiatry, University of Goettingen, Von-Siebold-Str. 5, D-37075 Goettingen, Germany

Department of Psychiatry and Psychotherapy, University of Erlangen, Schwabachanlage 6, D-91054 Erlangen, Germany

Behavioral and Brain Functions 1744-9081 2006 2 1 5 http://www.behavioralandbrainfunctions.com/content/2/1/5 16441883 10.1186/1744-9081-2-5 26 10 2005 27 1 2006 27 1 2006 2006 Veit et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The objective of this study was to analyze the extracellularly acting semicarbazide-sensitive amine oxidase (SSAO) serum levels in children with ADHD for the first time. SSAO is known to show deviations from normal in various somatic disorders and to interplay with the intracellularly active MAO. In humans two forms of SSAO a circulating form in plasma and a membrane-bound form are involved in monoaminergic metabolism.

Methods

We analyzed serum levels of SSAO in 27 children meeting ICD-10 criteria of Hyperkinetic Disorder (F90) or DSM-IV criteria of ADHD combined type by HPLC method and fluorimetric detection. A group of 42 healthy volunteers within the same age range (7.0 – 14.0 years) served as controls.

Results

No significant differences between children with ADHD (SSAO activity M = 773, SD = 217 mU/l) and healthy controls (SSAO activity M = 775, SD = 256 mU/l) in SSAO serum levels were found (F = 2.18; p > 0.14). Further, stimulant medication status had no influence on the result (F = 2.52; p > 0.11).

Conclusion

There is no evidence for a deviation of SSAO serum activity in ADHD. Hence, extracellularly acting SSAO does not seem to be a promising factor for further research in ADHD. But progress in knowledge of its physiologic role and of the relationship between the membrane-bound and the circulating serum form may open new avenues for research on SSAO in ADHD.

Background

There is increasing evidence that deviations of the dopaminergic, noradrenergic, and serotonergic systems are involved in attention deficit hyperactivity disorder (ADHD) 1. Several biogenic amines of these systems are degraded by oxidative deamination via the intracellular enzyme group monoamine oxidases (MAO). Thus the latter play an important role concerning the modification of signal transduction within these neurotransmitter systems 2.

In children with ADHD, significantly lower levels of platelet MAO activity seem to be associated with increased impulsivity and inattention 3. In addition, different medications with MAO inhibiting properties have been shown to be effective in the pharmacological treatment of ADHD (for a review see 45). Further, MAO seems to be a good candidate for genetic investigations of ADHD since DNA variations in this gene may play a role in the predisposition to the disorder. However, studies showed mixed results 6789 and focusing on the intracellularly acting MAO only seems to be a too narrow look.

Other aminoxidases like the semicarbacide sensitive amino oxidase (SSAO) may also be involved in ADHD. Since SSAO acts extracellularly, it may work complementary to the intracellular activity of MAO 10. Additionally, there is direct physiological interplay between the activities of SSAO and MAO 11.

In humans two forms of SSAO have been detected: a circulating form in plasma and a membrane-bound form. Both forms of SSAO metabolize several aromatic and aliphatic primary amines including dopamine while generating at the same time hydrogen peroxide and ammonia. The presence of SSAO in human cerebrovascular tissues and in endothelial cells from microvessels (including the blood-brain barrier) 1213 (for a review see14) supports its impact on CNS metabolism and makes it a candidate to be investigated in ADHD.

Therefore, to shed more light on this issue our pilot-study analyzed the extracellularly acting SSAO serum levels in ADHD for the first time.

Methods and materials

Patients

A total of 27 children meeting ICD-10 criteria of Hyperkinetic Disorder (F90) or DSM-IV criteria of ADHD combined type (assessment by clinical interview, physical examination and Diagnostic Checklist, DCL-HKS 15) (age range 7.0 – 14.0 years) were included. All were recruited sequentially from the outpatient clinic of the Department of Child and Adolescent Psychiatry of the University of Erlangen-Nuremberg (n = 12) and the University of Goettingen (n = 15). Further inclusion criteria were the following: intelligence IQ ≥ 85, body weight > 20 kg. Exclusion criteria were a clinical diagnosis of a developmental disorder or psychosis, depressive disorder, anxiety disorder, tic disorder, substance abuse, previous or recent epilepsy, EEG registration indicating epileptiform activity, previous/recent internal medicine or other neurological problems or treatment with known SSAO-inhibitory substances (i.e. tricyclic antidepressants) or other antidepressants. 13 children received methylphenidate, 14 were medication free.

A group of 42 healthy volunteers within the same age range (7.0 – 14.0 years) served as controls.

In view of the fact that SSAO circulating in plasma is quite stable 16 only one blood sample was collected from each participant. Because in SSAO activity no sex differences and no effect of body position (standing, sitting, supine) as well as of time of day of blood sampling has been observed1718, we did not control for these conditions.

This pilot study was carried out with the approval of the local Ethics Committees of the Medical Faculties of the Universities of Erlangen-Nuremberg and Goettingen. Written informed consent has been obtained from all parents and the patients able to write; younger children gave their oral assent.

Laboratory methods

The SSAO activity was determined according to a method of van Dijk et al. 19, modified in a few points. Initially, the enzymes MAO-A and MAO-B were inhibited in a 30-minute pre-incubation with chlorgyline, followed by conversion of benzylamine to benzaldehyde catalyzed by SSAO at 37°C over 60 minutes.

After precipitation of the proteins contained in the reaction mixture and stopping of the reaction, the benzaldehyde formed was derivatized with 5,5-dimethyl-1,3-cyclohexandione (dimedone) from the supernatant, which specifically reacts with aldehydes. The derived material was quantified using an isocratic HPLC method and fluorimetric detection (λ excitation 386 nm, λ emission 451 nm). For evaluation of the chromatograms, the software ChromGate, Version 2.8 (Knauer) was used.

In the present study, in contrast to the method of van Dijk et al. 19, serum was used as sample material instead of plasma. In addition, semicarbazide was used as an inhibitor for the sample blanks, since the MDL-72,145A used by van Dijk et al. 19 does not have any particular advantages over semicarbazide and the inhibition of the SSAO with semicarbazide is better established in the literature. Semicarbazide does not interfere either with the chromatogram or with the derivatization reaction of benzaldehyde.

In several tests, it was established that, by acidification with hydrochloric acid instead of sulfuric acid, and a pH range thus set at between 4.0 and 5.1, the derivatization was completed within 45 min and thus produced maximal measurement signals.

The problem described by van Dijk et al. 19 that the excess pressure developed in the reaction vessels by the temperature increase caused the lids to blow off if a hole was not made in the lids, was avoided here by using threaded vessels with a rubber seal (Sarstedt).

Statistics

Statistical analysis was performed using univariate analysis of variance to test the effect of group and medication status. The analyses were carried out using SPSS statistical software (SPSS, release 10.0.5 for Windows).

Results

In controls and ADHD children the SSAO serum activity (Fig. 1) was within the range of the sample of Boomsma et al. 16 with 147 healthy children and adolescents (about 620 ± 250 mU/l). The somewhat lower SSAO activity in the latter study, compared to the values presented here, might be due to age effects with older children in Boomsma et al. 16.

Figure 1

SSAO serum activity (mU/l) in children with ADHD and controls

SSAO serum activity (mU/l) in children with ADHD and controls.

The univariate analysis of variance revealed no differences between children with ADHD (SSAO activity M = 773, SD = 217 mU/l) and healthy children (SSAO activity M = 775, SD = 256 mU/l) (F = 0.86; p > 0.36; η_p²= 0.01; df = 1) and no influence of stimulant medication status (F = 2.52; p > 0.12; η_p²= 0.04; df = 1).

Discussion

The present study investigated the first time extracellularly acting SSAO in children with ADHD to test for its possible role within the neurobiological background of this disorder. No significant differences between children with ADHD and healthy controls in SSAO serum levels were found. Further, stimulant medication status had no influence on the result.

The finding is in line with normal SSAO levels in spontaneously hypertensive rats (SHR; an animal model of ADHD) 202122 and questions a differentiating influence of SSAO in ADHD.

Reduced quality of measurement underlying the lack of differences could be excluded due to our findings of stable SSAO serum activity in healthy adults (two blood samples per person, three hours apart) 23. But several limitations might contribute to the absence of found group differences.

In human brain the existence of SSAO is still under consideration 14. Due to its selective, membrane-bound expression in human brain blood vessels, but not in brain parenchyma (i.e. neurons and glial cells) 1213 SSAO has been related to the blood-brain barrier in addition to the better known, intracellularly acting MAO 24. Both metabolize circulating amines to maintain brain homeostasis. The origin of the circulating serum SSAO (analyzed in the present study) and its relationship to the membrane-bound form is still a matter of debate 16. It seems to be released mainly from vascular endothelial cells 2526, adipocytes 27 and to some part from skeleton 28. However at present it is only justifiable to examine serum SSAO, because the examination of the membrane-bound form in humans is restricted to highly invasive techniques of tissue extraction. Thus, the present analysis of SSAO serum activity reflects only one portion of the SSAO system possibly involved in monoaminergic abnormalities in ADHD.

The small effect sizes of aminoxidase differences found in mental disorders, e.g. MAO in schizophrenia 29, might also be present in SSAO and thus contribute to the absence of SSAO differences in our small sample. However, alterations in SSAO activity observed in disease states are generally more dramatic than those reported for MAO 10. But we detected only small effects according to Cohen 30 concerning the absence of both group differences of SSAO serum activity and of influence of medication status. Nevertheless, there exists some, albeit equivocal, evidence for an ADHD specific alteration concerning catechol-O-methyltransferase (COMT) gene and MAO A and B genes as well as MAO serum levels (e.g. lower levels of platelet MAO activity in untreated children and adolescents with ADHD 3).

Thus our negative finding concerning group differences of SSAO serum activity has to be interpreted with some caution because it is difficult to be certain to what extent it may reflect a lack of statistical power to detect small effect sizes.

Conclusion

In sum, there is no evidence for a deviation of SSAO serum activity in ADHD. Hence, extracellularly acting SSAO does not seem to be a promising factor for further research in ADHD. But progress in knowledge of its physiologic role and of the relationship between the membrane-bound and the circulating serum form may open new avenues for research on SSAO in ADHD.

List of abbreviations

semicarbazide-sensitive amine oxidase (SSAO)

attention deficit hyperactivity disorder (ADHD)

monoamine oxidases (MAO)

high pressure liquid chromatography (HPLC)

electroencephalography (EEG)

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

VR conceived of the study, participated in its design, recruited half of the patients and drafted the manuscript. HU recruited half of the patients and participated in study coordination. AB performed the statistical analysis. GB carried out the laboratory analyses and helped to draft the manuscript (methods). SB conceived of the study, participated in its design and coordination. AR participated in study coordination and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

ADHD - beyond core symptoms Rothenberger A Döpfner M Sergeant S Steinhausen HC Eur Child Adolesc Psychiatry 2004 13 Suppl 1 Monoamine oxidase in neuropsychiatry and behavior Shih JC Thompson RF Am J Hum Genet 1999 65 593 598 10.1086/302562 10441564 Platelet MAO and measures of attention and impulsivity in boys with attention deficit disorder and hyperactivity Shekim WO Bylund DB Alexson J Glaser RD Jones SB Hodges K Perdue S Psychiatry Res 1986 18 179 188 10.1016/0165-1781(86)90029-6 3725999 Non-stimulant medications in the treatment of ADHD Banaschewski T Roessner V Dittmann RW Santosh PJ Rothenberger A Eur Child Adolesc Psychiatry 2004 13 Suppl 1 I102 16 15322961 The safety of non-stimulant agents for the treatment of attention-deficit hyperactivity disorder Himpel S Banaschewski T Heise CA Rothenberger A Expert Opin Drug Saf 2005 4 311 321 10.1517/14740338.4.2.311 15794722 Association analysis of the monoamine oxidase A and B genes with attention deficit hyperactivity disorder (ADHD) in an Irish sample: preferential transmission of the MAO-A 941G allele to affected children Domschke K Sheehan K Lowe N Kirley A Mullins C O'Sullivan R Freitag C Becker T Conroy J Fitzgerald M Gill M Hawi Z Am J Med Genet B Neuropsychiatr Genet 2005 134 110 114 15717295 Association analysis of monoamine oxidase A and attention deficit hyperactivity disorder Lawson DC Turic D Langley K Pay HM Govan CF Norton N Hamshere ML Owen MJ O'Donovan MC Thapar A Am J Med Genet B Neuropsychiatr Genet 2003 116 84 89 10.1002/ajmg.b.10002 12497620 Family-based and association studies of monoamine oxidase A and attention deficit hyperactivity disorder (ADHD): preferential transmission of the long promoter-region repeat and its association with impaired performance on a continuous performance test (TOVA) Manor I Tyano S Mel E Eisenberg J Bachner-Melman R Kotler M Ebstein RP Mol Psychiatry 2002 7 626 632 10.1038/sj.mp.4001037 12140786 Linkage studies between attention-deficit hyperactivity disorder and the monoamine oxidase genes Jiang S Xin R Lin S Qian Y Tang G Wang D Wu X Am J Med Genet 2001 105 783 788 10.1002/ajmg.10098 11803531 Monoamine oxidases: certainties and uncertainties Tipton KF Boyce S O'Sullivan J Davey GP Healy J Curr Med Chem 2004 11 1965 1982 15279561 Inhibition of monoamine oxidase modulates the behaviour of semicarbazide-sensitive amine oxidase (SSAO) Fitzgerald DH Tipton KF J Neural Transm 2002 109 251 265 10.1007/s007020200021 11956949 Presence of SSAO in human and bovine meninges and microvessels Castillo V Lizcano JM Unzeta M Neurobiology (Bp) 1999 7 263 272 10689384 Semicarbazide-sensitive amine oxidase (SSAO) from human and bovine cerebrovascular tissues: biochemical and immunohistological characterization Castillo V Lizcano JM Visa J Unzeta M Neurochem Int 1998 33 415 423 10.1016/S0197-0186(98)00045-X 9874092 Semicarbazide-sensitive amine oxidase (SSAO) in the brain Obata T Neurochem Res 2002 27 263 268 10.1023/A:1014911209925 11958526 Diagnostik-System für Psychische Störungen im Kindes- und Jugendalter nach ICD-10 und DSM-IV (DISYPS-KJ) Döpfner M Lehmkuhl G Bern, Huber 2 2000 Semicarbazide-sensitive amine oxidase (SSAO): from cell to circulation Boomsma F Hut H Bagghoe U van der Houwen A van den Meiracker A Med Sci Monit 2005 11 RA122 126 15795708 Semicarbazide-sensitive amine oxidase: current status and perspectives Matyus P Dajka-Halasz B Foldi A Haider N Barlocco D Magyar K Curr Med Chem 2004 11 1285 1298 15134520 Plasma semicarbazide-sensitive amine oxidase in human (patho)physiology Boomsma F Bhaggoe UM van der Houwen AM van den Meiracker AH Biochim Biophys Acta 2003 1647 48 54 12686107 Determination of semicarbazide-sensitive amine oxidase activity in human plasma by high-performance liquid chromatography with fluorimetric detection van Dijk J Boomsma F Alberts G Man in 't Veld AJ Schalekamp MA J Chromatogr B Biomed Appl 1995 663 43 50 10.1016/0378-4347(94)00408-W 7704212 Monoamine oxidase and semicarbazide-sensitive amine oxidase activities in isolated cardiomyocytes of spontaneously hypertensive rats Pino R Failli P Mazzetti L Buffoni F Biochem Mol Med 1997 62 188 196 10.1006/bmme.1997.2633 9441872 Monoamine oxidase and semicarbazide-sensitive amine oxidase in spontaneously hypertensive and in normotensive control rats Guffroy C Strolin Benedetti M Life Sci 1984 34 535 545 10.1016/0024-3205(84)90486-7 6694535 Animal models of attention-deficit hyperactivity disorder Russell VA Sagvolden T Johansen EB Behav Brain Funct 2005 1 9 1180819 16022733 10.1186/1744-9081-1-9 Decreased SSAO serum levels in patients with major depression Roessner V Weber A Beck G Frieling H Bleich S Progress in Neuro-Psychopharmacology & Biological Psychiatry submitted Some properties of semicarbazide-sensitive amine oxidase (SSAO) from human cerebrovascular tissues Smeraldi C Castillo V Lizcano JM Unzeta M Inflamm Res 2001 50 Suppl 2 S144 5 11411595 Origins of serum semicarbazide-sensitive amine oxidase Stolen CM Yegutkin GG Kurkijarvi R Bono P Alitalo K Jalkanen S Circ Res 2004 95 50 57 10.1161/01.RES.0000134630.68877.2F 15178639 In vitro expression of benzylamine oxidase activity in cultured porcine smooth muscle cells Hysmith RM Boor PJ J Cardiovasc Pharmacol 1987 9 668 674 2442532 The release of soluble VAP-1/SSAO by 3T3-L1 adipocytes is stimulated by isoproterenol and low concentrations of TNFalpha Garcia-Vicente S Abella A Viguerie N Ros-Baro A Camps M Testar X Palacin M Zorzano A Marti L J Physiol Biochem 2005 61 395 401 16180338 Is semicarbazide-sensitive amine oxidase in blood plasma partly derived from the skeleton? Ekblom J Gronvall JL Garpenstrand H Nillson S Oreland L Neurobiology (Bp) 2000 8 129 135 11061210 Platelet monoamine oxidase in schizophrenia: a meta-analysis Marcolin MA Davis JM Schizophr Res 1992 7 249 267 10.1016/0920-9964(92)90019-2 1356426 Statistical Power Analysis for the Behavioral Sciences Cohen J Hillsdale, NJ, Erlbaum 2nd ed 1988