Publications by authors named "Henk van Lenthe"

29 Publications

  • Page 1 of 1

Comparison of the Diagnostic Performance of C26:0-Lysophosphatidylcholine and Very Long-Chain Fatty Acids Analysis for Peroxisomal Disorders.

Front Cell Dev Biol 2020 29;8:690. Epub 2020 Jul 29.

Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam UMC, Amsterdam Gastroenterology and Metabolism, University of Amsterdam, Amsterdam, Netherlands.

Peroxisomes are subcellular organelles that are involved in various important physiological processes such as the oxidation of fatty acids and the biosynthesis of bile acids and plasmalogens. The gold standard in the diagnostic work-up for patients with peroxisomal disorders is the analysis of very long-chain fatty acid (VLCFA) levels in plasma. Alternatively, C26:0-lysophosphatidylcholine (C26:0-LPC) can be measured in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS); a fast and easy method but not yet widely used. Currently, little is known about the correlation of C26:0-LPC in DBS and C26:0-LPC in plasma, and how C26:0-LPC analysis compares to VLCFA analysis in diagnostic performance. We investigated the correlation between C26:0-LPC levels measured in DBS and plasma prepared from the same blood sample. For this analysis we included 43 controls and 38 adrenoleukodystrophy (ALD) (21 males and 17 females) and 33 Zellweger spectrum disorder (ZSD) patients. In combined control and patient samples there was a strong positive correlation between DBS C26:0-LPC and plasma C26:0-LPC, with a Spearman's rank correlation coefficient of (114) = 0.962, < 0.001. These data show that both plasma and DBS are suitable to determine blood C26:0-LPC levels and that there is a strong correlation between C26:0-LPC levels in both matrices. Following this, we investigated how VLCFA and C26:0-LPC analysis compare in diagnostic performance for 67 controls, 26 ALD males, 19 ALD females, and 35 ZSD patients. For C26:0-LPC, all ALD and ZSD samples had C26:0-LPC levels above the upper limit of the reference range. For C26:0, one out of 67 controls had C26:0 levels above the upper reference range. For 1 out of 26 (1/26) ALD males, 1/19 ALD females and 3/35 ZSD patients, the C26:0 concentration was within the reference range. The C26:0/C22:0 ratio was within the reference range for 0/26 ALD males, 1/19 ALD females and 2/35 ZSD patients. Overall, these data demonstrate that C26:0-LPC analysis has a superior diagnostic performance compared to VLCFA analysis (C26:0 and C26:0/C22:0 ratio) in all patient groups. Based on our results we recommend implementation of C26:0-LPC analysis in DBS and/or plasma in the diagnostic work-up for peroxisomal disorders.
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http://dx.doi.org/10.3389/fcell.2020.00690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438929PMC
July 2020

Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor.

Front Cell Dev Biol 2020 17;8:499. Epub 2020 Jun 17.

Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam UMC, Amsterdam Gastroenterology and Metabolism, University of Amsterdam, Amsterdam, Netherlands.

X-linked adrenoleukodystrophy (ALD) is a devastating metabolic disorder affecting the adrenal glands, brain and spinal cord. Males with ALD are at high risk for developing adrenal insufficiency or progressive cerebral white matter lesions (cerebral ALD) at an early age. If untreated, cerebral ALD is often fatal. Women with ALD are not at risk for adrenal insufficiency or cerebral ALD. Newborn screening for ALD in males enables prospective monitoring and timely therapeutic intervention, thereby preventing irreparable damage and saving lives. The Dutch Ministry of Health adopted the advice of the Dutch Health Council to add a boys-only screen for ALD to the newborn screening panel. The recommendation made by the Dutch Health Council to only screen boys, without gathering any unsolicited findings, posed a challenge. We were invited to set up a prospective pilot study that became known as the SCAN study (SCreening for ALD in the Netherlands). The objectives of the SCAN study are: (1) designing a boys-only screening algorithm that identifies males with ALD and without unsolicited findings; (2) integrating this algorithm into the structure of the Dutch newborn screening program without harming the current newborn screening; (3) assessing the practical and ethical implications of screening only boys for ALD; and (4) setting up a comprehensive follow-up that is both patient- and parent-friendly. We successfully developed and validated a screening algorithm that can be integrated into the Dutch newborn screening program. The core of this algorithm is the "X-counter." The X-counter determines the number of X chromosomes without assessing the presence of a Y chromosome. The X-counter is integrated as second tier in our 4-tier screening algorithm. Furthermore, we ensured that our screening algorithm does not result in unsolicited findings. Finally, we developed a patient- and parent-friendly, multidisciplinary, centralized follow-up protocol. Our boys-only ALD screening algorithm offers a solution for countries that encounter similar ethical considerations, for ALD as well as for other X-linked diseases. For ALD, this alternative boys-only screening algorithm may result in a more rapid inclusion of ALD in newborn screening programs worldwide.
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http://dx.doi.org/10.3389/fcell.2020.00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311642PMC
June 2020

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

Brain 2019 11;142(11):3382-3397

Manchester Centre for Genomics Medicine, St Mary's Hospital, Manchester University Hospital Foundation Trust, Health Innovation Manchester, Oxford Road, Manchester, UK.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.
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http://dx.doi.org/10.1093/brain/awz291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821184PMC
November 2019

A mutation creating an upstream translation initiation codon in SLC22A5 5'UTR is a frequent cause of primary carnitine deficiency.

Hum Mutat 2019 10 3;40(10):1899-1904. Epub 2019 Jul 3.

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology and Metabolism Research Institute, University of Amsterdam, Amsterdam, The Netherlands.

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.
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http://dx.doi.org/10.1002/humu.23839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790604PMC
October 2019

Comparison of C26:0-carnitine and C26:0-lysophosphatidylcholine as diagnostic markers in dried blood spots from newborns and patients with adrenoleukodystrophy.

Mol Genet Metab 2017 12 28;122(4):209-215. Epub 2017 Oct 28.

Laboratory Genetic Metabolic Diseases, Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Pediatric Neurology, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy with a birth incidence of 1:14,700 live births. The disease is caused by mutations in ABCD1 and characterized by very long-chain fatty acids (VLCFA) accumulation. In childhood, male patients are at high-risk to develop adrenal insufficiency and/or cerebral demyelination. Timely diagnosis is essential. Untreated adrenal insufficiency can be life-threatening and hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. For this reason, ALD is being added to an increasing number of newborn screening programs. ALD newborn screening involves the quantification of C26:0-lysoPC in dried blood spots which requires a dedicated method. C26:0-carnitine, that was recently identified as a potential new biomarker for ALD, has the advantage that it can be added as one more analyte to the routine analysis of amino acids and acylcarnitines already in use. The first objective of this study was a comparison of the sensitivity of C26:0-carnitine and C26:0-lysoPC in dried blood spots from control and ALD newborns both in a case-control study and in newborns included in the New York State screening program. While C26:0-lysoPC was elevated in all ALD newborns, C26:0-carnitine was elevated only in 83%. Therefore, C26:0-carnitine is not a suitable biomarker to use in ALD newborn screen. In women with ALD, plasma VLCFA analysis results in a false negative result in approximately 15-20% of cases. The second objective of this study was to compare plasma VLCFA analysis with C26:0-carnitine and C26:0-lysoPC in dried blood spots of women with ALD. Our results show that C26:0-lysoPC was elevated in dried blood spots from all women with ALD, including from those with normal plasma C26:0 levels. This shows that C26:0-lysoPC is a better and more accurate biomarker for ALD than plasma VLCFA levels. We recommend that C26:0-lysoPC be added to the routine biochemical array of diagnostic tests for peroxisomal disorders.
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http://dx.doi.org/10.1016/j.ymgme.2017.10.012DOI Listing
December 2017

Evaluation of C26:0-lysophosphatidylcholine and C26:0-carnitine as diagnostic markers for Zellweger spectrum disorders.

J Inherit Metab Dis 2017 11 4;40(6):875-881. Epub 2017 Jul 4.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.

Introduction: Zellweger spectrum disorders (ZSD) are a group of genetic metabolic disorders caused by a defect in peroxisome biogenesis. This results in multiple metabolic abnormalities, including elevated very long-chain fatty acid (VLCFA) levels. Elevated levels of C26:0-lysophosphatidylcholine (C26:0-lysoPC) have been shown in dried blood spots (DBS) from ZSD patients. However, little is known about the sensitivity and specificity of this marker and C26:0-carnitine, another VLCFA-marker, in ZSD. We investigated C26:0-lysoPC and C26:0-carnitine as diagnostic markers for ZSD in DBS and fibroblasts.

Methods: C26:0-lysoPC levels in 91 DBS from 37 different ZSD patients were determined and compared to the levels in 209 control DBS. C26:0-carnitine levels were measured in 41 DBS from 29 ZSD patients and 97 control DBS. We measured C26:0-lysoPC levels in fibroblasts from 24 ZSD patients and 61 control individuals.

Results: Elevated C26:0-lysoPC levels (>72 nmol/L) were found in 86/91 ZSD DBS (n=33/37 patients) corresponding to a sensitivity of 89.2%. Median level was 567 nmol/l (range 28-3133 nmol/l). Consistently elevated C26:0-carnitine levels (>0.077 μmol/L) in DBS were found in 16 out of 29 ZSD patients corresponding to a sensitivity of 55.2%. C26:0-lysoPC levels were elevated in 21/24 ZSD fibroblast lines.

Discussion: C26:0-lysoPC in DBS is a sensitive and useful marker for VLCFA accumulation in patients with a ZSD. C26:0-carnitine in DBS is elevated in some ZSD patients, but is less useful as a diagnostic marker. Implementation of C26:0-lysoPC measurement in the diagnostic work-up when suspecting a ZSD is advised. This marker has the potential to be used for newborn screening for ZSD.
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http://dx.doi.org/10.1007/s10545-017-0064-0DOI Listing
November 2017

Identification and diagnostic value of phytanoyl- and pristanoyl-carnitine in plasma from patients with peroxisomal disorders.

Mol Genet Metab 2017 07 6;121(3):279-282. Epub 2017 May 6.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, 1105, AZ, The Netherlands.

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 phytanoyl- and 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.
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http://dx.doi.org/10.1016/j.ymgme.2017.05.003DOI Listing
July 2017

A sensitive mass spectrometry platform identifies metabolic changes of life history traits in C. elegans.

Sci Rep 2017 05 25;7(1):2408. Epub 2017 May 25.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands.

Abnormal nutrient metabolism is a hallmark of aging, and the underlying genetic and nutritional framework is rapidly being uncovered, particularly using C. elegans as a model. However, the direct metabolic consequences of perturbations in life history of C. elegans remain to be clarified. Based on recent advances in the metabolomics field, we optimized and validated a sensitive mass spectrometry (MS) platform for identification of major metabolite classes in worms and applied it to study age and diet related changes. Using this platform that allowed detection of over 600 metabolites in a sample of 2500 worms, we observed marked changes in fatty acids, amino acids and phospholipids during worm life history, which were independent from the germ-line. Worms underwent a striking shift in lipid metabolism after early adulthood that was at least partly controlled by the metabolic regulator AAK-2/AMPK. Most amino acids peaked during development, except aspartic acid and glycine, which accumulated in aged worms. Dietary intervention also influenced worm metabolite profiles and the regulation was highly specific depending on the metabolite class. Altogether, these MS-based methods are powerful tools to perform worm metabolomics for aging and metabolism-oriented studies.
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http://dx.doi.org/10.1038/s41598-017-02539-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445081PMC
May 2017

C26:0-Carnitine Is a New Biomarker for X-Linked Adrenoleukodystrophy in Mice and Man.

PLoS One 2016 28;11(4):e0154597. Epub 2016 Apr 28.

Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849772PMC
April 2017

A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses.

PLoS One 2015 25;10(9):e0138622. Epub 2015 Sep 25.

Department of Pediatric Metabolic Diseases, Emma Children's Hospital and Amsterdam Lysosome Center 'Sphinx', Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Introduction: Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III).

Methods: Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs.

Results: The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them.

Conclusion: The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138622PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583541PMC
May 2016

Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy.

Mol Genet Metab 2015 Feb 29;114(2):129-37. Epub 2014 Oct 29.

Stem Cell & Neurotherapies, Centre for Genomic Medicine, University of Manchester, Manchester, UK.

Background: Antibody formation can interfere with effects of enzyme replacement therapy (ERT) in lysosomal storage diseases. Biomarkers are used as surrogate marker for disease burden in MPS I, but large systematic studies evaluating the response of biomarkers to ERT are lacking. We, for the first time, investigated the response of a large panel of biomarkers to long term ERT in MPS I patients and correlate these responses with antibody formation and antibody mediated cellular uptake inhibition.

Methods: A total of 428 blood and urine samples were collected during long-term ERT in 24 MPS I patients and an extensive set of biomarkers was analyzed, including heparan sulfate (HS) and dermatan sulfate (DS) derived disaccharides; total urinary GAGs (DMBu); urinary DS:CS ratio and serum heparin co-factor II thrombin levels (HCII-T). IgG antibody titers and the effect of antibodies on cellular uptake of the enzyme were determined for 23 patients.

Results: Median follow-up was 2.3 years. In blood, HS reached normal levels more frequently than DS (50% vs 12.5%, p=0.001), though normalization could take several years. DMBu normalized more rapidly than disaccharide levels in urine (p=0.02). Nineteen patients (83%) developed high antibody titers. Significant antibody-mediated inhibition of enzyme uptake was observed in 8 patients (35%), and this correlated strongly with a poorer biomarker response for HS and DS in blood and urine as well as for DMBu, DS:CS-ratio and HCII-T (all p<0.006).

Conclusions: This study shows that, despite a response of all studied biomarkers to initiation of ERT, some biomarkers were less responsive than others, suggesting residual disease activity. In addition, the correlation of cellular uptake inhibitory antibodies with a decreased biomarker response demonstrates a functional role of these antibodies which may have important clinical consequences.
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http://dx.doi.org/10.1016/j.ymgme.2014.10.012DOI Listing
February 2015

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism.

Biochim Biophys Acta 2013 Jun 24;1832(6):773-9. Epub 2013 Feb 24.

Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal.

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.
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http://dx.doi.org/10.1016/j.bbadis.2013.02.012DOI Listing
June 2013

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long-term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans.

J Inherit Metab Dis 2013 Mar 19;36(2):247-55. Epub 2012 Sep 19.

Department of Pediatrics, Amsterdam Lysosome Centre Sphinx, Academic Medical Center, University Hospital of Amsterdam, Amsterdam, The Netherlands.

Introduction: Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels.

Methods: Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test.

Results: The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state.

Conclusions: HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.
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http://dx.doi.org/10.1007/s10545-012-9538-2DOI Listing
March 2013

Functional redundancy of mitochondrial enoyl-CoA isomerases in the oxidation of unsaturated fatty acids.

FASEB J 2012 Oct 10;26(10):4316-26. Epub 2012 Jul 10.

Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases (F0-222), Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.
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http://dx.doi.org/10.1096/fj.12-206326DOI Listing
October 2012

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling.

Biochim Biophys Acta 2010 Sep 9;1802(9):728-32. Epub 2010 Jun 9.

Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8 microM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.
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http://dx.doi.org/10.1016/j.bbadis.2010.06.002DOI Listing
September 2010

Supernatant of stored platelets causes lung inflammation and coagulopathy in a novel in vivo transfusion model.

Blood 2010 Aug 17;116(8):1360-8. Epub 2010 May 17.

Department of Intensive Care Medicine, Academic Medical Center, Amsterdam.

Transfusion-related acute lung injury is suggested to be a "2-hit" event resulting from priming and activation of pulmonary neutrophils. Activation may result from infusion of lysophosphatidylcholines (LysoPCs), which accumulate during storage of blood products. In the present study, we developed a syngeneic in vivo transfusion model to test whether storage of platelet concentrates (PLTs) results in lung injury in healthy rats as well as in a "2-hit" model using lipopolysaccharide-pretreated rats. In addition, the effect of washing of platelets was studied. In healthy rats, transfusion of aged PLTs caused mild lung inflammation. In LPS-pretreated rats, transfusion of aged PLTs, but not fresh PLTs, augmented pulmonary systemic coagulopathy. When PLTs components were transfused separately, supernatant of aged PLTs, but not washed aged platelets, induced pulmonary injury in the "2-hit" model. Supernatants of aged PLTs contained increased concentrations of LysoPCs compared with fresh PLTs, which enhanced neutrophil priming activity in vitro. We conclude that transfusion of aged PLTs induces lung inflammation in healthy rats. In a "2-hit" model, aged PLTs contribute to pulmonary and systemic coagulopathy, which may be mediated by LysoPCs, which accumulate in the supernatant of PLTs during storage.
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http://dx.doi.org/10.1182/blood-2009-10-248732DOI Listing
August 2010

Cardiolipin and monolysocardiolipin analysis in fibroblasts, lymphocytes, and tissues using high-performance liquid chromatography-mass spectrometry as a diagnostic test for Barth syndrome.

Anal Biochem 2009 Apr 31;387(2):230-7. Epub 2009 Jan 31.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.

Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the tafazzin (or TAZ) gene and is clinically characterized by (cardio)myopathy, neutropenia, and growth abnormalities. Biochemical abnormalities include decreased levels of the mitochondrial phospholipid cardiolipin, increased levels of monolysocardiolipin, and a lower degree of unsaturation of the (monolyso)cardiolipin acyl chains. Diagnostic testing for BTHS is routinely performed by TAZ gene sequencing, and recently a BTHS screening method in bloodspots has been developed, but both methods have important limitations. Because a validated confirmatory method is not yet available, we set up and validated a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for BTHS in cultured fibroblasts, lymphocytes, and skeletal muscle based on cardiolipin, monolysocardiolipin, and the monolysocardiolipin/cardiolipin ratio. In addition, we performed retrospective analysis of 121 muscle samples of patients with myopathy of which mitochondrial origin was presumed, and we identified one patient with cardiolipin abnormalities similar to BTHS patients. Molecular analysis revealed a bona fide mutation in the TAZ gene. We conclude that (monolyso)cardiolipin analysis by HPLC-MS not only is a powerful tool to diagnose patients with clinical signs and symptoms of BTHS but also should be used in patients suffering from mitochondrial myopathies with unknown etiology.
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http://dx.doi.org/10.1016/j.ab.2009.01.032DOI Listing
April 2009

Quantitative analysis of the experimental cytotoxic drug cyclopentenyl cytosine and its metabolite in plasma with HPLC tandem mass spectrometry.

Biomed Chromatogr 2008 Dec;22(12):1368-73

Leiden University Medical Center, Department of Clinical Pharmacy and Toxicology, Leiden, The Netherlands.

The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by HPLC-electrospray ionization tandem mass spectrometry. The calibration curves for CPEC and the metabolite CPEU were linear up to 10 microm with correlation coefficients of 0.997 (SD = 0.002, n = 10) and 0.997 (SD = 0.004, n = 10), respectively. The detection limit for CPEC was 0.03 and 0.12 microM for CPEU. The intra- and interassay coefficients of variation for CPEC and CPEU were less then 10%. The usefulness of the method was demonstrated by analyzing CPEC and CPEU in plasma of a patient treated with CPEC. HPLC with electrospray ionization tandem mass spectrometry allowed rapid and sensitive determination of CPEC and CPEU levels in plasma.
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http://dx.doi.org/10.1002/bmc.1067DOI Listing
December 2008

Bloodspot assay using HPLC-tandem mass spectrometry for detection of Barth syndrome.

Clin Chem 2008 Feb 10;54(2):371-8. Epub 2007 Dec 10.

Department of Clinical Chemistry, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands.

Background: Barth syndrome (BTHS) is a serious X-linked, metabolic, multisystem disorder characterized by cardiomyopathy, neutropenia, myopathy, and growth delay. Because early diagnosis and appropriate treatment are of key importance for the survival of affected boys, we developed a biochemical BTHS screening method based on analysis of the monolysocardiolipin:cardiolipin ratio in bloodspots.

Methods: We performed chloroform/methanol extraction on quarter-inch punches of dried bloodspots on Guthrie cards from BTHS patients and controls. Extracts were dried (60 degrees C, N(2)) and reconstituted in CHCl(3)/methanol/H(2)O [50:45:5 vol/vol/vol, 0.1% NH(3) (25%)]. HPLC-tandem mass spectrometry analysis was performed with a normal-phase HPLC column and multiple reaction monitoring transitions for monolysocardiolipin (MLCL) and cardiolipin (CL) with a total run time of 10 min. The ratio of MLCL and CL was used as screening parameter.

Results: All BTHS patients (n = 31) had monolysocardiolipin:cardiolipin ratios >0.40 and all controls (n = 215) had monolysocardiolipin:cardiolipin ratios <0.23. Using a cutoff point of 0.30, a blind test of 206 samples (199 controls, 7 BTHS) had sensitivity and specificity of 100%. Bloodspots could be stored at 4 degrees C or room temperature for >1 year without affecting the test outcome. Three neonatal Guthrie cards of BTHS patients taken 3.6 to 5.8 years previously were correctly identified as positive for BTHS.

Conclusions: HPLC-tandem mass spectrometry analysis of dried bloodspots is an unambiguous screening test for BTHS with potential for rapid screening of neonates suspected of having BTHS, making remote and retrospective diagnosis accessible for a disease that is almost certainly underdiagnosed.
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http://dx.doi.org/10.1373/clinchem.2007.095711DOI Listing
February 2008

HPLC-electrospray tandem mass spectrometry for rapid determination of dihydropyrimidine dehydrogenase activity.

Clin Chem 2007 Mar 1;53(3):528-30. Epub 2007 Feb 1.

Emma Children's Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Background: Patients with a partial dihydropyrimidine dehydrogenase (DPD) deficiency have an increased risk of developing severe 5-fluorouracil-associated toxicity. We developed a rapid and specific method to measure the DPD activity in peripheral blood mononuclear cells using HPLC tandem-mass spectrometry (HPLC-MS/MS).

Methods: The activity of DPD was measured with thymine as the substrate, followed by reversed-phase HPLC combined with electrospray ionization MS/MS and detection of the product dihydrothymine with multiple-reaction monitoring. Stable-isotope labeled dihydrothymine was used as the internal standard.

Results: Dihydrothymine was measured within an analytical run of 10 min, with a lower limit of quantification of 54 microg/L (0.4 micromol/L). The intraassay and interassay variations of the DPD activity assay were both <7%. A linear correlation (R(2) = 0.980; P <0.001) was observed between the HPLC-MS/MS data and those obtained with a reference method using radiolabeled thymine. There were no systematic differences between the 2 methods, and both methods yielded similar results.

Conclusion: The analysis of the DPD activity with HPLC-MS/MS is rapid, accurate, and sufficiently sensitive to be used as a screening method for patients with a DPD deficiency.
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http://dx.doi.org/10.1373/clinchem.2006.074096DOI Listing
March 2007

Increased dihydropyrimidine dehydrogenase activity associated with mild toxicity in patients treated with 5-fluorouracil and leucovorin.

Eur J Cancer 2007 Jan 13;43(2):459-65. Epub 2006 Nov 13.

Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Department of Clinical Chemistry, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands.

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5FU. The prognostic significance of DPD activity in peripheral blood mononuclear (PBM) cells and buccal mucosa cells with respect to toxicity was investigated in 44 patients treated with 5FU-leucovorin. Grade III/IV haematological and grade III/IV gastrointestinal toxicity were observed in 25% and 21% of the patients, respectively. No association was observed between the DPD activity in buccal mucosa cells and toxicity. In contrast, the mean DPD activity in PBM cells proved to be increased in patients experiencing grade I/II neutropenia when compared to patients without neutropenia and those suffering from grade III/IV neutropenia (P=0.002). Patients with a high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-leucovorin, suggesting an important role of DPD in the aetiology of toxicity associated with catabolites of 5FU.
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http://dx.doi.org/10.1016/j.ejca.2006.09.017DOI Listing
January 2007

Identification and characterization of human cardiolipin synthase.

FEBS Lett 2006 May 27;580(13):3059-64. Epub 2006 Apr 27.

Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, University of Amsterdam, Academic Medical Center, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands.

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.
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http://dx.doi.org/10.1016/j.febslet.2006.04.054DOI Listing
May 2006

Preparation, radiochemical purity control and stability of 99mTc-mertiatide (Mag-3).

Ann Nucl Med 2005 Jun;19(4):345-9

Department of Nuclear Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

Background: Scintigraphic image analysis of 99mTc-mertiatide (Mag-3, mercaptoacetyltriglycine) clearance provides the determination of the blood flow, the tubular transit time and the excretion as well from both kidneys. Radiopharmaceutical routine recommends a radiochemical purity control before administration of the product to a patient. The main objective of this study is to develop a Mag-3 labeling procedure that fits better than the previous one in our daily routine production of radiopharmaceuticals.

Methods: Increasing proportions of 99mTc-Mag-3 were measured during the heating and cooling steps of the Mag-3 labeling procedure. HPLC analysis was used to confirm the results of a rapid radiochemical quality control assay on standard ITLC-SG paper.

Results: The reconstitution time takes 20-25 minutes from the harvest of pertechnetate to a ready-for-use calibrated patient syringe. The HPLC profile of 99mTc-Mag-3 including its minor impurities remains unchanged for 24-48 hours after reconstitution.

Conclusions: The application of a programmable Peltier-directed device for heating/cooling provides a better control of the temperature course. The procedure proposed fully meets the labeling criteria recommended by the supplier and can be performed with a minimum of attention within a time-span that we formerly needed for solely the radiochemical purity control assay. Moreover, 99mTc-Mag-3 prepared in this way seems to be considerably more stable than mentioned in the manufacturer's instructions.
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http://dx.doi.org/10.1007/BF02984631DOI Listing
June 2005

beta-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities.

Hum Mol Genet 2004 Nov 22;13(22):2793-801. Epub 2004 Sep 22.

Department of Clinical Chemistry, Emma Children's Hospital, The Netherlands.

beta-Ureidopropionase deficiency is an inborn error of the pyrimidine degradation pathway, affecting the cleavage of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid. In this study, we report the elucidation of the genetic basis underlying a beta-ureidopropionase deficiency in four patients presenting with neurological abnormalities and strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid in plasma, cerebrospinal fluid and urine. No beta-ureidopropionase activity could be detected in a liver biopsy obtained from one of the patients, which reflected the complete absence of the beta-ureidopropionase protein. Analysis of the beta-ureidopropionase gene (UPB1) of these patients revealed the presence of two splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). Heterologous expression of the mutant enzyme in Escherichia coli showed that the A85E mutation resulted in a mutant beta-ureidopropionase enzyme without residual activity. Our results demonstrate that the N-carbamyl-beta-amino aciduria in these patients is due to a deficiency of beta-ureidopropionase, which is caused by mutations in the UPB1 gene. Furthermore, an altered homeostasis of beta-aminoisobutyric acid and/or increased oxidative stress might contribute to some of the clinical abnormalities encountered in patients with a beta-ureidopropionase deficiency. An analysis of the presence of the two splice site mutations and the missense mutation in 95 controls identified one individual who proved to be heterozygous for the IVS8-1G>A mutation. Thus, a beta-ureidopropionase deficiency might not be as rare as is generally considered.
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http://dx.doi.org/10.1093/hmg/ddh303DOI Listing
November 2004

Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry.

Clin Chem 2004 Nov 16;50(11):2117-24. Epub 2004 Sep 16.

Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Departments of Clinical Chemistry, Amsterdam, The Netherlands.

Background: The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry.

Methods: Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards.

Results: All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls.

Conclusion: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.
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http://dx.doi.org/10.1373/clinchem.2004.038869DOI Listing
November 2004

Quantification of 5,6-dihydrouracil by HPLC-electrospray tandem mass spectrometry.

Clin Chem 2004 Jan;50(1):236-8

Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Department of Clinical Chemistry, PO Box 22700, 1100 DE Amsterdam, The Netherlands.

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http://dx.doi.org/10.1373/clinchem.2003.026229DOI Listing
January 2004

New insights in dihydropyrimidine dehydrogenase deficiency: a pivotal role for beta-aminoisobutyric acid?

Biochem J 2004 Apr;379(Pt 1):119-24

Emma Children's Hospital and Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands.

DPD (dihydropyrimidine dehydrogenase) constitutes the first step of the pyrimidine degradation pathway, in which the pyrimidine bases uracil and thymine are catabolized to beta-alanine and the R-enantiomer of beta-AIB (beta-aminoisobutyric acid) respectively. The S-enantiomer of beta-AIB is predominantly derived from the catabolism of valine. It has been suggested that an altered homoeostasis of beta-alanine underlies some of the clinical abnormalities encountered in patients with a DPD deficiency. In the present study, we demonstrated that only a slightly decreased concentration of beta-alanine was present in the urine and plasma, whereas normal levels of beta-alanine were present in the cerebrospinal fluid of patients with a DPD deficiency. Therefore the metabolism of beta-alanine-containing peptides, such as carnosine, may be an important factor involved in the homoeostasis of beta-alanine in patients with DPD deficiency. The mean concentration of beta-AIB was approx. 2-3-fold lower in cerebrospinal fluid and urine of patients with a DPD deficiency, when compared with controls. In contrast, strongly decreased levels (10-fold) of beta-AIB were present in the plasma of DPD patients. Our results demonstrate that, under pathological conditions, the catabolism of valine can result in the production of significant amounts of beta-AIB. Furthermore, the observation that the R-enantiomer of beta-AIB is abundantly present in the urine of DPD patients suggests that significant cross-over exists between the thymine and valine catabolic pathways.
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http://dx.doi.org/10.1042/BJ20031463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1224056PMC
April 2004

Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene.

J Gene Med 2003 Dec;5(12):1018-27

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic.

Methods: To study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus-mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post-infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria.

Results: We show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10-30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial-membrane potential, morphological abnormalities, and liver dysfunction.

Conclusions: These data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non-proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination.
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http://dx.doi.org/10.1002/jgm.450DOI Listing
December 2003