Technical and biochemical factors affecting cerebrospinal fluid 5-MTHF, biopterin and neopterin concentrations

doi:10.1016/j.ymgme.2008.07.004

Molecular Genetics and Metabolism

Volume 95, Issue 3, November 2008, Pages 127-132

https://doi.org/10.1016/j.ymgme.2008.07.004 Get rights and content

Abstract

Background: The diagnosis of pediatric neurologic disorders with a deficiency in the biosynthesis of either the neurotransmitters serotonin and dopamine, or the co-factor tetrahydrobiopterin or a cerebral 5-methyltetrahydrofolate (5-MTHF) deficiency, strongly relies on a robust analysis of neurotransmitter metabolites, pterins and 5-MTHF in the cerebrospinal fluid (CSF). The aim of this study was to investigate which technical and biochemical factors affect the CSF concentration of 5-MTHF, neopterin and biopterin in a pediatric population.

Methods: We studied effects of the ventriculo-spinal gradient, total protein concentration, pretreatment with ascorbic acid (in case of 5-MTHF analysis), pretreatment of CSF with trichloro acetic acid (TCA)/dithiotreitol (DTE) and oxidation with either iodine or manganese oxide (in case of pterin analysis), storage time and age of the patients. We included CSF samples from children until the age of 18 years and analysed 5-MTHF, neopterin, biopterin, homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA) and total protein.

Results: The major findings of our study are: (1) CSF 5-MTHF, neopterin and biopterin concentrations are not affected by the ventriculo-spinal gradient; (2) pretreatment of CSF with ascorbic acid has negligible effects on 5-MTHF concentrations; (3) pretreatment of CSF with TCA/DTE and oxidation with iodine results in the most accurate determination of neopterin and biopterin; (4) when adjusted for age and total protein, CSF 5-MTHF correlated with 5-HIAA, but not with HVA; (5) the reference value of 5-MTHF in CSF in childhood is age-dependent (r = −0.634; p ⩽ 0.001); (6) we did not observe an age-dependency for neopterin and biopterin in CSF.

Conclusion: 5-MTHF, neopterin and biopterin can be analysed in any volume of CSF that is collected. For correct analysis of pterins, CSF will have to be pretreated to stabilize the concentrations and stored properly, whereas such pretreatment is not necessary for 5-MTHF.

Introduction

Several pediatric neurological disorders are characterized by a decreased production of the neurotransmitters serotonin, dopamine and (nor)epinephrin (Fig. 1). Some of these disorders are caused by a defect in the biosynthesis of these neurotransmitters, e.g. deficiencies in tyrosine hydroxylase (TH) [1] or aromatic aminoacid decarboxylase (AADC) [2]. These disorders are characterized by strongly reduced cerebrospinal fluid (CSF) concentrations of homovanillic acid (HVA) in case of TH deficiency or of HVA and 5-hydroxy-indoleacetic acid (5-HIAA) in case of AADC deficiency. Other disorders are caused by a defect in the biosynthesis of an essential co-factor, tetrahydrobiopterin (BH₄). BH₄ is, amongst others, a co-factor for TH, tryptophan hydroxylase (TPH) and phenylalanine hydroxylase (PAH). A deficiency in BH₄ synthesis leads to reduced activity of these enzymes or the accumulation of inhibitors of the enzymes, such as dihydrobiopterin (BH₂). BH₄ is synthesized from guanosine triphosphate (GTP) by the subsequent action of various enzymes (Fig. 2). Mutations in several of these enzymes (including GTP cyclohydrolase (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR) and dihydropteridine reductase (DHPR)) may each lead to an aberrant synthesis of BH₄ and each of these disorders is characterized by a specific pattern of neopterin, biopterin and sepiapterin in the CSF [3].

In the central nervous system, folate is predominantly present as 5-methyltetra-hydrofolate (5-MTHF). A few years ago, a neurological syndrome designated as cerebral folate deficiency (CFD) has been described. CFD is biochemically characterized by low CSF 5-MTHF concentrations with normal concentrations in plasma [4]. In addition to (primary) CFD, a number of disorders are associated with a secondary decrease in cerebral folate, such as Rett syndrome, Aicardi–Goutières syndrome and Kearns–Sayre syndrome [5], [6], [7], [8].

The diagnosis of the above-mentioned neurological disorders have in common that they rely on the analysis of CSF [9]. In general, the analysis of HVA, 5-HIAA, neopterin, biopterin, sepiapterin and 5-MTHF will provide a characteristic biochemical profile that leads to the correct diagnosis. The importance of a correct and robust analysis of CSF is substantiated by the fact that some of these disorders can be very well treated, such as l-dopa treatment in TH, SR and GTPCH deficiencies, and folinic acid treatment in CFD. In a number of previous publications, the technical requirements for a correct analysis of HVA and 5-HIAA have been described, including the need for collection of a standardized volume of CSF and the application of age-dependent reference values [1]. In contrast, however, the pre-analytical and analytical requirements for a robust analysis of neopterin, biopterin and 5-MTHF have not been thoroughly studied.

The aim of this study was to investigate some of the technical and biochemical factors that may affect the CSF concentration of 5-MTHF, neopterin and biopterin, such as sample pretreatment and the effect of the ventriculo-spinal gradient, as well as to define the age-dependency of reference values for these compounds in a pediatric population.

Section snippets

Patients

This study was approved by the local medical ethical committee. We included CSF samples from children until the age of 18 years, who underwent a lumbar puncture mainly for evaluation of developmental delay, seizures or suspected intracranial infection. Patients did not suffer from an inborn error of folate metabolism, tetrahydrobiopterin biosynthesis, neurotransmitter metabolism or any other known inborm error of metabolism. Lumbar punctures were performed at the Department of Pediatric

5-MTHF

We did not observe differences in the 5-MTHF concentrations in the three successive fractions of CSF that were analysed in the 25 patients (p = 0.99; ANOVA test). However, we observed a small, but significant, difference in 5-MTHF concentrations measured in either the ascorbic acid-treated or -untreated samples (p < 0.0001, paired-samples t-test, n = 36). Levels were a mean 4.7% lower in the untreated than in the ascorbic acid-treated samples. Furthermore, we did not observe a difference between the

Discussion

The major findings of our study are: (1) CSF 5-MTHF, neopterin and biopterin concentrations are not affected by the ventriculo-spinal gradient, at least as far as the first 9 ml CSF obtained by lumbar puncture are involved; (2) pretreatment of CSF with ascorbic acid only has marginal effects on 5-MTHF concentrations; (3) pretreatment of CSF with TCA/DTE followed by oxidation with I₂ results in the most accurate preservation and analysis of CSF for determination of neopterin and biopterin; (4)

Acknowledgments

This work was supported by grants Zon-MW Innovational Research (No. 917.46.331, “Vidi program”, to M.M.V.). We thank Gerard Jorna and Ben Geurtz for technical assistance.

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Cerebral folate deficiency in adults: A heterogeneous potentially treatable condition
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To describe the phenotype and the response to folinic acid supplementation of cerebral folate deficiency (CFD) in adults, a disorder diagnosed on low 5-methyltetrahydro-folate (5MTHF) in cerebrospinal fluid (CSF), which can correspond to a inherited disorder of folate metabolism (IDFM) or to a metabolic consequence of various neurological diseases.
We conducted a retrospective study on 224 adult patients with neurological symptoms who had a 5MTHF CSF dosage, collecting their neurologic and neuroimaging data.
69 patients had CFD (CSF 5MTHF level < 41 nmol/L), 25 of them had severe CFD (sCFD; ≤25 nmol/L) with adult onset neurological symptoms in 41%. 56% of sCFD patients had an underlying identified neurologic disorder, mainly mitochondrial diseases, hepatic encephalopathy and primary brain calcifications (no identified IDFM), the others were classified as undiagnosed. sCFD patients presented most frequently pyramidal syndrome (75%), movement disorders (56%), cerebellar syndrome (50%) and intellectual disability (46%). MRI findings mostly showed white matter abnormalities (WMA; 32%) and calcifications (12%), and were normal in 23%. The clinico-radiological phenotype of sCFD patients was not clearly different from non CFD patients in terms of manifestations frequency. However, their neurological picture was more complex with a higher number of combined neurological symptoms (4.7±1.6 vs 3.4±1.7, p = .01). In Magnetic Resonance Spectroscopy (MRS), Choline/Creatine (Cho/Cr) ratio was lower in sCFD patients (n = 7) compared to non-CFD patients (n = 73) (p = .005), with good sensitivity (71%) and excellent specificity (92%). Among twenty-one CFD patients treated with folinic acid, nine had a sustained improvement, all with sCFD but one (50% of sCFD patients improved). In two undiagnosed patients with extremely low 5MTHF CSF values, MRI WMA and low Cho/Cr ratios, folinic acid treatment leaded to a dramatic clinical and radiological improvement.
CSF 5MTHF dosage should be considered in patients with mitochondrial diseases, primary brain calcifications and unexplained complex neurological disorders especially if associated with WMA, since folinic acid supplementation in patients with sCFD is frequently efficient.
Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1
2018, Blood Advances
Citation Excerpt :
In this case, even when the peak blood folate level was 2242 nM, the CSF 5-methyltHF concentration was 1/50th at 43 nM. Although CSF 5-methylTHF levels of 40 to 50 nM are sufficient in adults, this is far lower than the much higher CSF 5-methylTHF levels in heathy infants and toddlers.36,37 Hence, the choroid plexus represents a very tight barrier for folates in the absence of either PCFT or FRα.
Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V_max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates. The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.
Methods for assessment of folate (vitamin B<inf>9</inf>)
2018, Laboratory Assessment of Vitamin Status
Folate (vitamin B9) is a generic name for all derivatives of pteroylglutamic acid which differ from each other in the oxidation state, one carbon substituents, and number of polyglutames residues. Folates function as cofactors in the transfer and utilization of one carbon groups which play essential roles in several major cellular processes, including nucleic acid biosynthesis, amino acid metabolism, and methyl group biogenesis. These reactions are essential for the metabolism of homocysteine and the synthesis of purines and pyrimidines; hence in folate deficiency, the cell loses its ability to synthesize DNA and RNA and to divide. A deficiency of folate leads to megaloblastic anemia and is also the most common reason for raised plasma homocysteine. The risk of neural tube defects (NTDs), certain types of cancers, Alzheimer’s and cardiovascular disease increases in folate insufficiency. Serum and red cell folate (RCF) concentrations are the most frequent markers used to assess folate status. Total plasma homocysteine (tHcy) measurements are recommended to confirm folate insufficiency/deficiency. A variety of laboratory methods exist to measure the concentration of these markers.
Simultaneous measurement of monoamine metabolites and 5-methyltetrahydrofolate in the cerebrospinal fluid of children
2017, Clinica Chimica Acta
Citation Excerpt :
CFD has been increasingly recognized and 5-MTHF measurement is now among the routine assays performed at neurotransmitter laboratories [10]. Since there is no rostrocaudal gradient of 5-MTHF, any volume of CSF can be used for the 5-MTHF assay [10]. Monoamine metabolite assays have been conducted mainly by high-performance liquid chromatography (HPLC) with electrochemical detection [6,11–13].
We describe a new method for simultaneous measurement of monoamine metabolites (3-O-methyldopa [3-OMD], 3-methoxy-4-hydroxyphenylethyleneglycol [MHPG], 5-hydroxyindoleacetic acid [5-HIAA], and homovanillic acid [HVA]) and 5-methyltetrahydrofolate (5-MTHF) and its use on cerebrospinal fluid (CSF) samples from pediatric patients.
Monoamine metabolites and 5-MTHF were measured by high-performance liquid chromatography with fluorescence detection. CSF samples were prospectively collected from children according to a standardized collection protocol in which the first 1-ml fraction was used for analysis.
Monoamine metabolites and 5-MTHF were separated within 10 min. They showed linearity from the limit of detection to 1024 nmol/l. The limit of quantification of each metabolite was sufficiently low for the CSF sample assay. In 42 CSF samples after excluding cases with possibly altered neurotransmitter profiles, the concentrations of 3-OMD, MHPG, 5-HIAA, HVA, and 5-MTHF showed significant age dependence and their ranges were comparable with the reference values in the literature. The metabolite profiles of aromatic l-amino acid decarboxylase deficiency, Segawa disease, and folate receptor α defect by this method were compatible with those in the literature.
This method is a simple means of measuring CSF monoamine metabolites and 5-MTHF, and is especially useful for laboratories not equipped with electrochemical detectors.
The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption
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The major challenge is achieving adequate CSF folate and this requires careful monitoring CSFlevels until a satisfactory concentration is reached. Of importance is that normal CSF folate blood levels during infancy and early childhood are much higher than previously appreciated: ∼100–150 nM over the first year decreasing to ∼50–90 by five years, falling further to >60 nM during puberty (Ormazabal et al., 2011; Perez-Duenas et al., 2011; Verbeek et al., 2008). These levels are difficult to achieve in subjects with HFM even with extraordinarily high doses of parenteral folate (Torres et al., 2015).
The proton-coupled folate transporter (PCFT-SLC46A1) is the mechanism by which folates are absorbed across the brush-border membrane of the small intestine. The transporter is also expressed in the choroid plexus and is required for transport of folates into the cerebrospinal fluid. Loss of PCFT function, as occurs in the autosomal recessive disorder “hereditary folate malabsorption” (HFM), results in a syndrome characterized by severe systemic and cerebral folate deficiency. Folate-receptor alpha (FRα) is expressed in the choroid plexus, and loss of function of this protein, as also occurs in an autosomal recessive disorder, results solely in “cerebral folate deficiency” (CFD), the designation for this disorder. This paper reviews the current understanding of the functional and structural properties and regulation of PCFT, an electrogenic proton symporter, and contrasts PCFT properties with those of the reduced folate carrier (RFC), an organic anion antiporter, that is the major route of folate transport to systemic tissues. The clinical characteristics of HFM and its treatment, based upon the thirty-seven known cases with the clinical syndrome, of which thirty have been verified by genotype, are presented. The ways in which PCFT and FRα might interact at the level of the choroid plexus such that each is required for folate transport from blood to cerebrospinal fluid are considered along with the different clinical presentations of HFM and CFD.
Biochemical Analyses of Cerebrospinal Fluid for the Diagnosis of Neurometabolic Conditions. What Can We Expect?
2016, Seminars in Pediatric Neurology
In this article, we review the state-of-the-art analysis of different biomarkers in the cerebrospinal fluid for the diagnosis of genetically conditioned, rare, neurometabolic diseases, including glucose transport defects, neurotransmitter (dopamine, serotonin, and gamma-aminobutyric acid) and pterin deficiencies, and vitamin defects (folate, vitamin B₆, and thiamine) that affect the brain. The analysis of several key metabolites are detailed, which thus highlights the preanalytical and analytical factors that should be cautiously controlled to avoid misdiagnosis; moreover, these factors may facilitate an adequate interpretation of the biochemical profiles in the context of severe neuropediatric disorders. Secondary disturbances in these biomarkers, which are associated with other genetic or environmental conditions, are also detailed. Importantly, the early biochemical identification of biochemical disturbances in the cerebrospinal fluid may improve the clinical outcomes of a remarkable number of patients, who may exhibit good neurologic outcomes using the available therapies for these disorders.

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Technical and biochemical factors affecting cerebrospinal fluid 5-MTHF, biopterin and neopterin concentrations

Abstract

Introduction

Section snippets

Patients

5-MTHF

Discussion

Acknowledgments

Clin. Chim. Acta

J. Chromatogr.

J. Chromatogr.

J. Neurosci. Methods

Biochemical hallmarks of tyrosine hydroxylase deficiency

Clin. Chem.

Aromatic l-amino acid decarboxylase deficiency: clinical features treatment and prognosis

Neurology

Disorders of tetrahydrobiopterin and related biogenic amines

Cerebral folate deficiency

Dev. Med. Child Neurol.

Reduced folate transport to the CNS in female Rett patients

Neurology