Metabolomics to study functional consequences in peroxisomal disorders Herzog, K.

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1 UvA-DARE (Digital Academic Repository) Metabolomics to study functional consequences in peroxisomal disorders Herzog, K. Link to publication Citation for published version (APA): Herzog, K. (2017). Metabolomics to study functional consequences in peroxisomal disorders General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 21 Aug 2018

2 6 CHAPTER Identification and diagnostic value of phytanoyl- and pristanoylcarnitine in plasma from patients with peroxisomal disorders 6 Katharina Herzog, Henk van Lenthe, Ronald J. A. Wanders, Frédéric M. Vaz, Hans R. Waterham, and Sacha Ferdinandusse Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam 1105 AZ, the Netherlands Molecular Genetics and Metabolism 2017 Jul;121(3):

3 Abstract Phytanic acid is a branched-chain fatty acid, the level of which is elevated in patients with a variety of peroxisomal disorders, including Refsum disease, and Rhizomelic chondrodysplasia punctata type 1 and 5. Elevated levels of both phytanic and pristanic acid are found in patients with Zellweger Spectrum Disorders, and pristanic acid is elevated in patients with α-methylacyl-coa racemase deficiency. For the diagnosis of peroxisomal disorders, a variety of metabolites can be measured in blood samples from suspected patients, including very long-chain fatty acids, phytanic and pristanic acid. Based on the fact that very long-chain fatty acylcarnitines are elevated in tissues and plasma from patients with certain peroxisomal disorders, we investigated whether phytanoyl- and pristanoyl-carnitine are also present in plasma from patients with different peroxisomal disorders. Our study shows that phytanoyland pristanoyl-carnitine are indeed present in plasma samples from patients with different types of peroxisomal disorders, but only when the total plasma levels of their corresponding fatty acids, phytanic acid and pristanic acid, are markedly elevated. We conclude that the measurement of phytanoyl- and pristanoyl-carnitine is not sensitive and specific enough to use these acylcarnitines as conclusive diagnostic markers for peroxisomal disorders. Keywords: peroxisome, phytanic acid, pristanic acid, acylcarnitine, Refsum disease 116

4 Introduction Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid, which is derived from dietary sources, including dairy products, fish and meat. In patients with Refsum disease, phytanic acid metabolism is disturbed due to mutations in the gene encoding phytanoyl-coa hydroxylase (PHYH) (Fig. 1) 1,2. This results in the impaired α-oxidation of phytanic acid, which subsequently accumulates in tissues and plasma of these patients 3. Phytanic acid levels are also elevated in patients with the peroxisomal disorder Rhizomelic chondrodysplasia punctata (RCDP) type 1 4 and type 5 5. These patients have mutations in the PEX7 and PEX5 gene, respectively, which code for two cytosolic receptor proteins involved in the import of PTS2-targeted proteins into peroxisomes 3. Furthermore, α-oxidation is impaired in patients with a Zellweger Spectrum Disorder (ZSD), due to mutations in any of 13 different PEX genes, causing a defect in peroxisome biogenesis 3. Phytanic acid is converted to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) via α-oxidation in the peroxisome and, subsequently, pristanic acid is further broken down via peroxisomal β-oxidation (Fig. 1) 1. Both phytanic and pristanic acid levels are elevated in ZSD patients depending on the diet and age of the patient. Elevated levels of pristanic acid are also found in plasma from patients with α-methylacyl-coa racemase (AMACR) deficiency. AMACR is required for the conversion of the R-isomer of pristanic acid into the S-isomer, which is the only isomer that can be broken down via β-oxidation in the peroxisome 6. 6 Acylcarnitine analysis is an important diagnostic tool especially for the detection of patients suffering from a defect in mitochondrial fatty acid oxidation and/or a defect in amino acid degradation 7. Acylcarnitines are formed from the corresponding acyl-coa esters by a variety of different carnitine acyltransferases 7. Abnormalities in acylcarnitine profiles, notably C26:0-acylcarnitine, have also been described in patients with peroxisomal disorders 8. Besides the implementation of several acylcarnitines as biomarkers into newborn screening programs for a number of inborn errors of metabolism 9, acylcarnitine metabolites have also been reported in untargeted metabolomics approaches 10. The use of highthroughput omics techniques is becoming increasingly important in clinical diagnostics, and untargeted metabolomic approaches using mass spectrometry have recently been proposed to be potentially useful in the screening for inborn errors of metabolism 10. Based on the finding that very long-chain fatty acylcarnitines are elevated in tissues and plasma from patients with certain peroxisomal disorders 8,11 13, we investigated whether phytanoyl-carnitine is present in plasma from patients with elevated phytanic acid levels, including patients with Refsum disease, RCDP type I and ZSD patients. In addition, we investigated whether pristanoyl-carnitine can be detected in plasma from patients with AMACR deficiency. 117

5 R,S-Pristanoyl-CoA R,S-Phytanoyl-CoA ABCD3 ABCD3 ABCD3 ABCD3 PHYH α-oxidation R,S-Pristanic acid AMACR R-Pristanoyl-CoA S-Pristanoyl-CoA ß-oxidation 4,8-DMN-CoA CROT peroxisome 4,8-DMN-carnitine Figure 1. Schematic overview of branched-chain fatty acid catabolism in peroxisomes. The branched-chain fatty acids phytanic and pristanic acid are transported into the peroxisome as Coenzyme A (CoA)-ester via the membrane protein ABC transporter D3 (ABCD3). Phytanic acid first undergoes α-oxidation, resulting in the formation of pristanic acid. In Refsum disease, the enzyme phytanoyl-coa 2-hydroxylase (PHYH) is defective, and α-oxidation is blocked. The CoA-ester of pristanic acid first needs to be converted from the (R)-configuration into its (S)-configuration by the enzyme α-methylacyl-coa racemase (AMACR) before entering the β-oxidation pathway. Pristanic acid then undergoes chain-shortening via three rounds of β-oxidation producing 4,8-dimethylnonanoyl- CoA (4,8-DMN-CoA). Carnitine octanoyltransferase (CROT) converts 4,8-DMN-CoA into the corresponding carnitine ester (4,8-DMNcarnitine), which is exported to the mitochondrion for further oxidation. Results We measured phytanoyl-carnitine and pristanoyl-carnitine levels in plasma from patients with different peroxisomal disorders. We included plasma samples from patients with Refsum disease (n=8), RCDP type 1 (n=8), ZSDs (n=11), AMACR deficiency (n=4), in addition to plasma samples from control individuals (n=15). We analysed the plasma samples for straight-chain acylcarnitines with an acyl-chain length of C16 and C20, and the branched-chain fatty acylcarnitines phytanoylcarnitine and pristanoyl-carnitine (Fig. 2, supplemental Table 1). Phytanoyl-and pristanoylcarnitine have the same parent ion as straight-chain C20- and C19-carnitine, respectively, but can be distinguished based on retention time (Fig. 2). Phytanoyl-carnitine levels were significantly elevated in plasma from patients with Refsum disease and patients with RCDP type 1 when compared to healthy control samples (Fig. 3A). In plasma from ZSD patients, phytanoyl-carnitine was only moderately elevated in a few patient samples when compared to healthy controls (Fig. 3A). In plasma samples from AMACR patients, we only detected low levels of phytanoyl-carnitine in one patient sample. When comparing the levels of phytanoyl-carnitine to the total phytanic acid levels determined in the same plasma samples by GC-MS, we found a good correlation between these metabolites (Fig. 3C). 118

6 Levels of pristanoyl-carnitine were significantly elevated in plasmas from AMACR patients, when compared to healthy control samples (Fig. 3B). In plasma samples from patients affected by one of the other peroxisomal disorders, however, only very low or even trace amounts of pristanoyl-carnitine were found. The levels of pristanoyl-carnitine did not correlate well with the total pristanic acid levels (Fig. 3D). Noteworthy, we detected phytanoyl-carnitine and pristanoyl-carnitine only when the levels of their corresponding fatty acids, phytanic acid and pristanic acid, were markedly elevated in the same plasma samples and exceeded the diagnostic reference values (phytanic acid: > 10.0 µm; pristanic acid: > 3.0 µm). In particular, the levels of phytanoyl-carnitine were detected in all samples with phytanic acid levels 60.0 µm, six times higher than the upper limit of the diagnostic reference range (supplemental Table 1, supplemental Fig. 1A). Similarly, increased levels of pristanoyl-carnitine were measured only when pristanic acid levels were 16.3 µm, more than five times higher than the upper limit of the diagnostic reference range (supplemental Table 1, supplemental Fig. 1B) > 85 relative abundance (% of highest peak) relative abundance (% of highest peak) Pristanoyl-carnitine 1.40 Phytanoyl-carnitine 1.77 C19-carnitine 2.38 C20-carnitine > > 85 6 relative abundance (% of highest peak) D3-C16-carnitine Time (min) Figure 2. Representative extracted ion chromatograms of newly identified acylcarnitine species. Transitions are shown for C20- and phytanoyl-carnitine, C19- and pristanoyl-carnitine, and the internal standard D3-C16-carnitine. 119

7 A A Phytanoyl-carnitine B B Pristanoyl-carnitine Refsum Refsum RCDP RCDP AMACR **** AMACR * ZSD ZSD **** Controls Controls µm µm C C Phytanic acid / phytanoyl-carnitine D D Pristanic acid / pristanoyl-carnitine phytanoyl-carnitine (µm) 0.3 R 2 = R 2 = r s = r s = phytanoyl-carnitine (µm) phytanic acid (µm) pristanic acid (µm) Figure 3. Concentrations and correlation of acylcarnitine and fatty acid levels. Box and whisker plot of phytanoyl-carnitine (A) and pristanoyl-carnitine (B) levels. Data are presented as mean ± SD. We performed one-way ANOVA to determine significant differences between the groups, compared to the healthy control group (**** p-value < ). Scatter plot of the correlation of phytanic acid (C) and pristanic acid (D) levels with the corresponding acylcarnitines. The coefficient of determination (R 2 ) was used as an estimate of goodness of fit of the linear regression model, and Spearman s rank correlation coefficient (r s ) was used as a non-parametric measure of correlation. 120

8 Discussion For the diagnosis of peroxisomal disorders, metabolite analysis is performed in patient material such as blood and urine upon suspicion of a peroxisomal defect based on the clinical presentation. Peroxisomal metabolites that should be measured are very long-chain acids (VLCFAs), phytanic and pristanic acid, bile acid intermediates di- and trihydroxycholestanoic acid, and pipecolic acid in plasma, plasmalogens in erythrocytes, and bile acid intermediates in urine 15. Recently, the determination of C26:0-lyso-PC and C26:0-carnitine in blood spots have been included in the standard diagnostic workflow for peroxisomal disorders 15. In this study we investigated whether measurement of phytanoyl- and pristanoylcarnitine using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) would be a valuable addition for the diagnostics of peroxisomal disorders. Elevated levels of phytanic acid in plasma and tissues are the biochemical hallmark for Refsum disease 1, and phytanic acid levels can also be elevated in RCDP type 1 and 5, and ZSDs. It is known that 4,8-dimethylnonanoyl-CoA, which is the end product of peroxisomal β-oxidation of pristanic acid, is converted to its carnitine ester by carnitine-octanoyl-transferase (CROT), and transported to the mitochondrion for further oxidation 16,17. Here we show that when the cell has to cope with elevated levels of phytanic or pristanic acid, these metabolites can also be converted to carnitine esters. This conversion is likely performed by CROT, as the enzyme has been described to accept branched-chain fatty acids as substrates 16, whereas phytanic and pristanic acid are not substrate for carnitine palmitoyltransferase 1 (CPT1) 18. Our findings are in agreement with elevated very long-chain fatty acylcarnitines reported in tissues and plasma from patients with peroxisomal disorders, in which increased levels of VLCFAs were reported 8, Compared to the standard method to measure phytanic and pristanic acid using GC-MS, measurement of phytanoyl- and pristanoyl-carnitine is considerably faster and requires no derivatization. In fact, sample preparation and the use of UPLC-MS/MS are similar to the method used for the determination of acylcarnitines, which is a commonly used assay in many metabolic laboratories. However, although the correlation between the phytanic acid and the phytanoyl-carnitine levels was good, the levels of phytanoyl-carnitine detected in plasma were very low when compared to total phytanic acid levels determined using GC- MS, and were elevated only when the levels of phytanic acid were markedly increased in the plasma samples. Elevated levels of pristanoyl-carnitine were only detected in patients with AMACR deficiency and two ZSD patients, but the levels detected in plasma also were very low, and the correlation of pristanoyl-carnitine to pristanic acid levels was poor. 121

9 In conclusion, we here report the identification of phytanoyl- and pristanoylcarnitine in plasma samples from patients with different types of peroxisomal disorders. Our study shows that when increased levels of phytanoyl- or pristanoyl-carnitines are found either by targeted acylcarnitine analysis, as presented in this paper, or in untargeted metabolomic approaches, the patient could be affected with a peroxisomal disorder and that follow-up biochemical testing is required using standard protocols for the diagnosis of a peroxisomal disorder. More importantly, our study shows that the measurement of phytanoyl- and pristanoyl-carnitine is not sensitive and specific enough for conclusive diagnosis of peroxisomal disorders, including Refsum disease and AMACR deficiency. Acknowledgements This work was supported by the FP-7-PEOPLE-2012-Marie Curie-ITN PERFUME (KH and HRW). The funding source had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 122

10 Materials and Methods Anonymised plasma samples from healthy individuals (n=15), and patients with Refsum disease (n=8), RCDP type 1 (n=8), ZSDs (n=11), and AMACR deficiency (n=4) were used for the determination of acylcarnitines. Twenty µl of plasma were mixed with 200 µl of internal standard (20 nm [16,16,16-2 H 3 ]-C16-carnitine (D3-C16-carnitine) in acetonitrile). Subsequently, samples were sonicated for 5 min in a water bath, followed by centrifugation for 5 min at 4 ᵒC with 20,000 g. The supernatant was evaporated at 60 ᵒC under a nitrogen stream, and the residue was dissolved in 50 µl methanol. Ten µl of extract were injected. Liquid chromatography was performed at 50 C using an Acquity BEH C18 column (Waters, Milford MA). Extracts were separated by a linear gradient between solution A (0.1% HCOOH in water) and solution B (100% methanol), with a gradient from 0 to 6 min from 20% solution A to 8% solution. The flow rate was 0.5 ml/min. A Micromass Quattro Premier XE Tandem Mass Spectrometer (Waters, Milford, MA) was used in the multiple reaction monitoring mode (MRM) in positive electrospray ionisation mode. The spray voltage used was 3.5 kv, source temperature was 130 ᵒC, and the desolvation temperature was 350 ᵒC. Cone gas flow: 50 L/hr, desolvation gas flow: 900 L/hr, collision gas pressure 2.5e-3 m bar. We determined a subset of acylcarnitine species, ranging from C16-carnitine to C20-carnitine, pristanoylcarnitine, and phytanoyl-carnitine. The common daughter ion was m/z Following parent ions were used for acylcarnitine species in MRM channels: m/z (D3-C16); m/z (C19; pristanoyl-carnitine); m/z (C20; phytanoyl-carnitine). Acylcarnitine levels were processed using Masslynx software. Phytanic acid and pristanic acid were measured using gas chromatography-mass spectrometry (GC-MS), as described previously 14. Data in figures are presented as mean ± SD, and one-way ANOVA was used for statistical comparison between the groups

11 References 1. Wanders, R. J. A., Komen, J. & Ferdinandusse, S. Phytanic acid metabolism in health and disease. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 1811, (2011). 2. Van Veldhoven, P. P. Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism. J. Lipid Res. 51, (2010). 3. Waterham, H. R., Ferdinandusse, S. & Wanders, R. J. A. Human disorders of peroxisome metabolism and biogenesis. Biochim. Biophys. Acta - Mol. Cell Res. 1863, (2016). 4. Braverman, N. E., Moser, A. B. & Steinberg, S. J. Rhizomelic Chondrodysplasia Punctata Type 1. GeneReviews( ) (University of Washington, Seattle, 1993). 5. Barøy, T. et al. A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform. Hum. Mol. Genet. 24, (2015). 6. Ferdinandusse, S. et al. Mutations in the gene encoding peroxisomal alpha-methylacyl-coa racemase cause adult-onset sensory motor neuropathy. Nat. Genet. 24, (2000). 7. Houten, S. M., Violante, S., Ventura, F. V & Wanders, R. J. A. The Biochemistry and Physiology of Mitochondrial Fatty Acid β-oxidation and Its Genetic Disorders. Annu. Rev. Physiol. 78, (2015). 8. Duranti, G. et al. Urine acylcarnitine analysis by ESI-MS/MS: A new tool for the diagnosis of peroxisomal biogenesis disorders. Clin. Chim. Acta 398, (2008). 9. Sandlers, Y. et al. Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: Prospective newborn screening for X-linked adrenoleukodystrophy. Mol. Genet. Metab. 105, (2012). 10. Miller, M. J. et al. Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism. J. Inherit. Metab. Dis. 38, (2015). 11. Van De Beek, M. C. et al. C26:0-Carnitine is a new biomarker for X-linked adrenoleukodystrophy in mice and man. PLoS One 11, 1 19 (2016). 12. Haynes, C. A. & De Jesús, V. R. Simultaneous quantitation of hexacosanoyl lysophosphatidylcholine, amino acids, acylcarnitines, and succinylacetone during FIA-ESI-MS/MS analysis of dried blood spot extracts for newborn screening. Clin. Biochem. 49, (2016). 13. Ferdinandusse, S. et al. Adult peroxisomal acyl-coenzyme A oxidase deficiency with cerebellar and brainstem atrophy. J. Neurol. Neurosurg. Psychiatry 81, (2010). 14. Vreken, P. et al. Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry. J. Chromatogr. B Biomed. Appl. 713, (1998). 15. Ferdinandusse, S., Ebberink, M. S., Vaz, F. M., Waterham, H. R. & Wanders, R. J. A. The important role of biochemical and functional studies in the diagnostics of peroxisomal disorders. J. Inherit. Metab. Dis. 39, (2016). 16. Ferdinandusse, S. et al. Molecular cloning and expression of human carnitine octanoyltransferase: evidence for its role in the peroxisomal beta-oxidation of branched-chain fatty acids. Biochem. Biophys. Res. Commun. 263, (1999). 17. Verhoeven, N. M. et al. Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts. J. Lipid Res. 39, (1998). 18. Singh, H. & Poulos, A. Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against??-oxidation of phytanic acid in rat liver mitochondria. FEBS Lett. 359, (1995). 124

12 Supplemental Files A A Phytanoyl-carnitine B B Pristanoyl-carnitine phytanoyl-carnitine (µm) pristanoyl-carnitine (µm) normal elevated normal elevated phytanic acid pristanic acid 6 Supplemental Figure 1. Phytanoyl- and pristanoyl-carnitine levels are only present in case of elevated levels of the free fatty acids. (A) Levels of phytanoyl-carnitine, and correlation of phytanoyl-carnitine to phytanic acid levels. Based on the upper limit of the reference range for phytanic acid (10.0 µm), results were depicted in either the normal or elevated group. (B) Levels of pristanoyl-carnitine, and correlation of pristanoyl-carnitine to pristanic acid levels. Based on the upper limit of the reference range for pristanic acid (3.0 µm), results were depicted in either the normal or elevated group. Supplemental Table 1. List of measured acylcarnitines ranging from C16 to C20, including the branched-chain acylcarnitines phytanoyl- and pristanoyl-carnitine. In addition, the measured concentrations of phytanic and pristanic acid as determined by GC-MS are listed per sample. Data were normalised to the internal standard, and data are presented in µm. Supplemental Table 1 can be found on the journal s website following this link: 125