Pentose phosphate pathway biochemistry, metabolism and inherited defects Amsterdam 2008 Mirjam M.C. Wamelink The research described in this thesis was carried out at the Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, The Netherlands. The publication of this thesis was financially supported by: Department of Clinical Chemistry, VU University Medical Center Amsterdam E.C. Noyons Stichting ter bevordering van de Klinische Chemie in Nederland J.E. Jurriaanse Stichting te Rotterdam Printed by: Printpartners Ipskamp BV, Enschede ISBN: 978-90-9023415-1 Cover: Representation of a pathway of sugar Copyright Mirjam Wamelink, Amsterdam, The Netherlands, 2008 2 VRIJE UNIVERSITEIT Pentose phosphate pathway biochemistry, metabolism and inherited defects ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L.M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Geneeskunde op donderdag 11 december 2008 om 13.45 uur in de aula van de universiteit, De Boelelaan 1105 door Mirjam Maria Catharina Wamelink geboren te Alkmaar 3 promotor: prof.dr.ir. C.A.J.M. Jakobs copromotor: dr. E.A. Struijs 4 Abbreviations 6PGD 6-phosphogluconate dehydrogenase ADP adenosine diphosphate ATP adenosine triphosphate CSF cerebrospinal fluid DHAP dihydroxyacetone phosphate G6PD glucose-6-phosphate dehydrogenase GA glyceraldehyde GAPDH glyceraldehyde-3-phosphate dehydrogenase GSG oxidized glutathione GSSG reduced glutathione LC-MS/MS liquid chromatography-tandem mass spectrometry MRI magnetic resonance imaging MRS magnetic resonance spectroscopy NAD(H) nicotinamide adenine dinucleotide (reduced) NADP(H) nicotinamide adenine dinucleotide phosphate (reduced) P phosphate(s) PPP pentose phosphate pathway ROS reactive oxygen species RPI ribose-5-phosphate isomerase TALDO transaldolase TPI triosephosphate isomerase 5 Contents Chapter 1 Outline of the thesis 7 Chapter 2 Quantification of sugar phosphate intermediates of the 13 pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism. J. Chromatogr. B, 823 (2005) 18–25. Chapter 3 Analysis of polyols in urine by liquid chromatography- 29 tandem mass spectrometry: a useful tool for recognition of inborn errors affecting polyol metabolism. J. Inherit. Metab. Dis. 28 (2005) 951–963. Chapter 4 Detection of Transaldolase Deficiency by Quantitation of 45 Novel Seven-Carbon Chain Carbohydrate Biomarkers in Urine. J. Inherit. Metab. Dis. 30 (2007) 735–742. Chapter 5 Retrospective detection of transaldolase deficiency in 59 amniotic fluid: Implications for prenatal diagnosis. Prenat. Diagn. 28 (2008) 460-462. Chapter 6 Transaldolase deficiency in a 2 year-old boy with 65 cirrhosis. Mol. Genet. Metab. 94 (2008) 255-258. Chapter 7 Sedoheptulokinase deficiency due to a 57-kb deletion in 73 cystinosis patients causes urinary accumulation of sedoheptulose: Elucidation of the CARKL gene. Hum. Mutat. 29 (2008) 532-536. Chapter 8 Dynamic rerouting of the carbohydrate flux is key to 85 counteracting oxidative stress. J. Biol. 6 (2007) 10 Chapter 9 Biochemistry, metabolism and inherited defects of the 117 pentose phosphate pathway: a review. J. Inherit. Metab. Dis. In press. Chapter 10 Summary and discussion 145 Nederlandse samenvatting 153 Dankwoord 160 Curriculum Vitae 162 Publications 163 6 Chapter 1 Chapter 1 OUTLINE OF THE THESIS 7 Chapter 1 Outline of the thesis The pentose phosphate pathway (PPP) consists of two parts, which fulfil two distinct roles: an oxidative, non-reversible limb which allows reduction of NADP+ to NADPH while converting glucose-6-phosphate to a pentose- phosphate and CO2, and a non-oxidative, reversible limb, which connects pentose-phosphates to glycolytic intermediates. In recent years, two defects in the PPP have been discovered [1-3]. The first defect is deficiency of ribose-5-phosphate isomerase (RPI, OMIM 608611) and has been diagnosed in one patient, who presented with a slowly progressive leukoencephalopathy. The other defect is deficiency of transaldolase (TALDO, OMIM 606003). This defect is associated with liver dysfunction, whereas other organs are affected in various degrees. These two defects are very rare and although the defected enzymes are present in the same metabolic pathway, the PPP, the clinical phenotypes of both diseases are completely different. The main research goals concerning this thesis were: To characterize the normal pattern of metabolites involved in the PPP (polyols, sugars and sugar-phosphates (sugar-P)) and to diagnose new patients with abnormal metabolite profiles with a defect in the PPP To elucidate further the clinical phenotype and pathology of patients with TALDO deficiency To investigate the function of the PPP and its interrelationship with connected pathways. This thesis is divided in three sections. The biochemical methods designed to study normal and abnormal metabolite patterns of intermediates in the PPP are described in chapters 2-4. The research performed to diagnose new TALDO deficient patients and further characterisation of the clinical phenotype is described in chapter 5 and 6. In the third section we studied the function and routing through the PPP (chapter 7 and 8). The PPP is an inter-conversion of sugar-P. In the oxidative part glucose-6- phosphate is converted to a pentose-phosphate which is converted in the non-oxidative part into other sugar-P. To investigate the intracellular concentrations of sugar-P in patients with a defect in the PPP a new method was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With this method described in chapter 2, the accumulation of sedoheptulose-7P in blood spots, fibroblasts and lymphoblasts of TALDO deficient patients could be identified. Furthermore, the LC-MS/MS method can be used for determining enzyme activities of 8 Chapter 1 the PPP by measuring the sugar-P involved as was done for TALDO and RPI deficiency. Polyols, or polyhydric alcohols, can be formed by the reduction of sugars and are classified based on the numbers of carbon (C) -atoms: erythritol and threitol (C4-polyols, tetritols), ribitol, arabitol and xylitol (C5-polyols, pentitols), galactitol, sorbitol and mannitol (C6-polyols, hexitols) and sedoheptitol and perseitol (C7-polyols, heptitols). Knowledge of the metabolism and function of most of the polyols is very limited. In TALDO deficiency elevated urinary concentrations of erythritol, arabitol and ribitol have been described [2]. In RPI deficiency arabitol and ribitol concentrations are highly elevated in urine, wheras xylitol is mildly elevated [1,3]. These two defects and other defects associated with the accumulation of polyols, like galactose-1-phosphate uridyltransferase deficiency, galactokinase deficiency, essential pentosuria and L- arabinosuria, can be diagnosed by the assessment of urinary concentrations of polyols. In chapter 3, two novel methods for the quantitative profiling of polyols in urine by LC-MS/MS are described. We started with the development of one method, which can be used as a quick screening method. The different polyol isomers elute as one peak and cannot be distinguished from each other. The second method uses the same sample preparation, however the use of another type of analytical liquid chromatography column makes separation of the different polyol isomers possible and thus enables the separate quantification of erythritol, threitol, ribitol, arabitol, xylitol, sorbitol, mannitol, galactitol, sedoheptitol and perseitol. In patients affected with TALDO deficiency, we previously also found increased amounts of a 7-carbon chain carbohydrate which we suspected of being sedoheptulose. In chapter 4 we describe the development of a LC-MS/MS method for identification and quantitation of the seven-carbon carbohydrates sedoheptulose and mannoheptulose in urine. Additionally, other seven-carbon chain carbohydrates were characterized in urine from controls and TALDO deficient patients, including sedoheptitol, perseitol and sedoheptulose-7P. The diagnosis of TALDO deficiency in a French family with four affected children born to the same consanguineous parents in 2006 expanded the number of TALDO deficient patients from two to six [4]. One of these patients presented in the antenatal period with hydrops fetalis with oligohydramnios. The pregnancy was medically terminated at 28 weeks gestation. In chapter 5 we retrospectively measured polyols, heptuloses and sedoheptulose-7P in the amniotic fluid sample of this fetus by LC- MS/MS. 9 Chapter 1 In chapter 6 we report the detailed clinical and biochemical presentation of a newly diagnosed patient with TALDO deficiency. As more patients are diagnosed, the clinical and biochemical phenotype of this disorder becomes more characterized and an overview is given in this chapter of all patients diagnosed together with the characteristics of the liver pathology. Little is known about the function of sedoheptulose and sedoheptulose-7P. Sedoheptulose-7P is one of the intermediates of the PPP and sedoheptulose and sedoheptulose-7P both accumulate in TALDO deficiency (chapter 2, 4-6). In chapter 7 we showed that patients with cystinosis caused by the common 57-kb deletion in the CTNS (cystinosin) gene excrete increased amounts of sedoheptulose in their urine. This 57-kb deletion also includes an adjacent gene CARKL. The CARKL gene encodes a protein that was predicted to function as a carbohydrate kinase.