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Analysis of Carnitine and Acylcarnitines in Biological Fluids and Application to a Clinical Study

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Analysis of Carnitine and Acylcarnitines in Biological Fluids and Application to a Clinical Study ANALYSIS OF CARNITINE AND ACYLCARNITINES IN BIOLOGICAL FLUIDS AND APPLICATION TO A CLINICAL STUDY Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Laurence Vernez aus Villars-Bramard, VD Genf, 2005 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von: Prof. Dr. Stephan Krähenbühl Prof. Dr. Gérard Hopfgartner Prof. Dr. Wolfgang Thormann Basel, den 6. April 2004 Prof. Dr. Marcel Tanner Dekan POUR TOI QUI M’ATTENDS ENCORE Un savant dans son laboratoire n’est pas seulement un technicien: c’est aussi un enfant placé en face de phénomènes naturels qui l’impressionnent comme un conte de fées Marie Curie-Sklodowska, 1933 Table of contents TABLE OF CONTENTS Résumé 13 Zusammenfassung 16 Summary 18 List of abbreviations 21 1. Introduction 23 1.1 General aspects 23 1.2 Carnitine functions 25 1.2.1. Mitochondrial long-chain fatty acid oxidation 25 1.2.2. Buffering of the mitochondrial acyl-CoA/CoA ratio 27 1.2.3. Removal of potentially toxic acyl-groups 27 1.2.4. Fatty acids oxidation in peroxisomes 28 1.3 Carnitine biosynthesis 28 1.4 Carnitine homeostasis 31 1.4.1. Absorption 31 1.4.2. Tissue distribution - carnitine transporters 32 i. Regulation of tissue distribution 33 ii. Kinetic of exogenous carnitine 33 1.4.3. Metabolism 34 1.4.4. Elimination - role of kidney 35 1.5 Carnitine deficiency 36 1.5.1. Primary carnitine deficiency 37 i. Systemic carnitine deficiency (SCD) 37 ii. Muscle carnitine deficiency (MCD) 38 1.5.2. Secondary carnitine deficiency 38 i. Metabolic disorders 39 ii. Acquired medical conditions 40 iii. Drug induced 40 iv. Hemodialysis treatment 41 1.6 Carnitine and acylcarnitines determination 45 1.6.1. Radioenzymatic assay 46 1.6.2. Gas chromatography 48 1.6.3. High-performance liquid chromatography 49 1.6.4. Capillary electrophoresis 51 1.6.5. Mass spectrometry 53 1.6.6. Carnitine extraction 57 1.7 Aims of the thesis 59 2. Analysis of carnitine and acylcarnitines in urine by capillary electrophoresis 63 2.1 Summary 64 2.2 Introduction 64 2.3 Experimental 66 2.3.1 Chemicals 66 2.3.2 Standard solutions 66 2.3.3 Sample preparation and derivatization 66 9 Table of contents 2.3.4 Capillary electrophoresis conditions 67 2.3.5 Radioenzymatic analysis of urine samples 67 2.4 Results and discussion 67 2.4.1 Choice of the internal standard 67 2.4.2 Separation of acylcarnitines 68 2.4.3 Method characterization 70 2.4.4 Application to urine samples 71 2.5 Conclusion 73 3. Determination of carnitine and acylcarnitines in urine by high-performance liquid chromatography – electrospray ionization ion trap tandem mass spectrometry 75 3.1 Summary 76 3.2 Introduction 76 3.3 Experimental 78 3.3.1 Chemicals 78 3.3.2 Preparation of internal standards and standard solutions 78 3.3.3 Urine sample collection 79 3.3.4 Urine sample preparation 79 3.3.5 HPLC-MS/MS analysis 80 3.3.6 Method validation 81 3.3.7 Urine 81 3.4 Results and discussion 81 3.4.1 Optimization of solid-phase extraction 82 3.4.2 Analysis of carnitine and acylcarnitines by HPLC-MS/MS 82 3.4.3 Method validation 85 3.4.4 Application to urine 87 3.5 Conclusion 89 4. Determination of carnitine and acylcarnitines in plasma by high-performance liquid chromatography – electrospray ionization ion trap tandem mass spectrometry 91 4.1 Summary 92 4.2 Introduction 92 4.3 Experimental 94 4.3.1 Chemicals 94 4.3.2 Instrumentation 94 4.3.3 Standard and internal standard solutions 94 4.3.4 Plasma sample collection 95 4.3.5 Sample preparation 95 4.3.6 Chromatographic conditions 96 4.3.7 Validation procedure 97 4.4 Results and discussion 97 4.4.1 Extraction procedure 97 4.4.2 HPLC-MS/MS separation 98 4.4.3 Matrix used for calibrators 98 4.4.4 Quantification in standard and plasma quality controls 100 4.4.5 Application to a serum sample from a patient with a metabolic disorder 102 4.5 Conclusion 104 10 Table of contents 5. Effect of L-carnitine supplementation on the kinetics of carnitine, acylcarnitines and butyrobetaine in patients with long-term hemodialysis 107 5.1 Summary 108 5.2 Introduction 108 5.3 Experimental 110 5.3.1 Study population 110 5.3.2 Study design 111 5.3.3 Steady state carnitine and acylcarnitines profiles between hemodialysis sessions 111 5.3.4 Carnitine and acylcarnitines kinetics during hemodialysis sessions (intra-dialysis) 112 5.3.5 Carnitine and acylcarnitines kinetics between two hemodialysis sessions (inter-dialysis) 112 5.3.6 Analysis of carnitine and acylcarnitines 112 5.3.7 Statistics and pharmacokinetic analysis 113 5.4 Results 114 5.4.1 Carnitine and acylcarnitines profiles 114 5.4.2 Intra-dialysis kinetics 116 5.4.3 Inter-dialysis kinetics 118 5.5 Discussion 120 6. Conclusion 125 References 129 Acknowledgments 149 Curriculum vitae 151 11 RRRRRR ÉÉÉÉÉÉ SSSSSS UUUUUU MMMMMM ÉÉÉÉÉÉ ZZZZZZ UUUUUU SSSSSS AAAAAA MMMMMM MMMMMM EEEEEE NNNNNN FFFFFF AAAAAA SSSSSS SSSSSS UUUUUU NNNNNN GGGGGG SSSSSS UUUUUU MMMMMM MMMMMM AAAAAA RRRRRR YYYYYY Résumé RÉSUMÉ La carnitine, un composé endogène présent dans la plupart des tissus animaux, est essentielle pour le transport des acides gras activés entre les organelles cellulaires et joue donc un rôle important dans le métabolisme des acides gras et dans la production d’énergie des cellules. La carnitine lie les acides gras et génère ainsi différentes acylcarnitines de longueurs de chaîne variables. La carnitine est également impliquée dans le maintien du réservoir cellulaire de coenzyme A libre et dans l’élimination d’acyl-CoAs potentiellement toxiques. Chez les mammifères, la carnitine est fournie pour deux tiers par un apport alimentaire et pour un tiers par biosynthèse à partir des acides aminés L-lysine et L- méthionine. Comme la carnitine est présente dans la plupart des tissus à des concentrations bien supérieures à celle du plasma, sa distribution depuis ses sites d’absorption et de synthèse à travers l’organisme est assurée par des systèmes de transport. Dans de nombreuses maladies métaboliques, le métabolisme de la carnitine est fortement perturbé, aboutissant à une redistribution du réservoir de carnitine et d’acylcarnitines. La détermination individuelle de chaque acylcarnitine dans les fluides biologiques est un procédé approprié pour diagnostiquer ces maladies. Le but de cette thèse était de développer des méthodes analytiques pour la détermination de la carnitine et des acylcarnitines dans les fluides biologiques. Finalement, une des méthodes développées a été utilisée pour le suivi d’une étude clinique. Les connaissances actuelles sur la carnitine et les acylcarnitines, notamment les fonctions de la carnitine, sa biosynthèse et son homéostasie, sont passées en revue dans le premier chapitre. Des cas de déficiences en carnitine sont également présentés et une description des différentes méthodes analytiques utilisées pour la détermination de la carnitine et des acylcarnitines complète cette partie introductive. Le chapitre 2 décrit une méthode d’électrophorèse capillaire développée pour déterminer le profil de la carnitine et des acylcarnitines à courte et moyenne chaîne, ceci après une extraction en phase solide sur une colonne de silice. Cet essai permet la séparation de la carnitine et de cinq acylcarnitines dans des solutions de standard, dans l’urine et dans des échantillons d’urine enrichie. Il a été validé pour la détermination de la carnitine et de l’acétylcarnitine dans des solutions de standard. Finalement, la carnitine a été quantifiée dans des échantillons d’urine et les résultats ont été comparés avec ceux obtenus par un essai radio-enzymatique. 13 Résumé Le chapitre 3 présente une méthode utilisant la chromatographie liquide à haute performance couplée à une détection par spectrométrie de masse en mode MS/MS (HPLC- MS/MS) pour la détermination de la carnitine et de huit différentes acylcarnitines, incluant les acylcarnitines à longue chaîne. Comme la détection est effectuée par spectrométrie de masse, une dérivatisation de la carnitine n’est pas nécessaire. Avant d’être injectés dans le système, les échantillons ont été soumis à une extraction en phase solide sur une colonne échangeuse de cations. La séparation à été obtenue en utilisant un réactif volatil de paires d’ions. La validation de la méthode pour la détermination de la carnitine dans des solutions de standard et des échantillons d’urine a été effectuée en utilisant un dérivé isotopique stable comme standard interne et de l’eau comme matrice de calibration. Les résultats obtenus pour la quantification de la carnitine dans des échantillons d’urine ont été comparés avec ceux issus d’une méthode radio-enzymatique. L’application à des échantillons d’urine provenant de patients souffrant de différentes aciduries organiques à permis de confirmer le diagnostic de ces maladies. L’extension de la méthode HPLC-MS/MS à des échantillons de plasma, après des modifications mineures du protocole d’extraction, notamment la précipitation des protéines, est rapportée dans le chapitre 4. La butyrobetaine (précurseur direct de la carnitine lors de sa biosynthèse) qui, au contraire de l’urine, est présente dans le plasma, a pu être déterminée avec la même méthode d’analyse. La quantification de la carnitine, de l’acétylcarnitine, de la propionylcarnitine, de l’isovalérylcarnitine, de l’hexanoylcarnitine, de l’octanoylcarnitine et de la butyrobetaine a été validée pour des solutions de standard et des échantillons de plasma en utilisant une solution aqueuse à 4% d’albumine sérique de bœuf comme matrice de calibration. Un échantillon de sérum d’un patient souffrant d’acidurie méthylmalonique a pu être identifié comme caractéristique de cette maladie.
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