Pterin determination in cerebrospinal fluid: state of the art

Introduction

Nowadays, pterins determination in cerebrospinal fluid (CSF) is routinely used for the investigation of inborn errors of neurotransmitters metabolism [1], [2], [3]. Inherited defects leading to neurotransmitter metabolism disorders have been described or predicted at the level of each enzyme involved in the synthesis of the neurotransmitters dopamine, (nor)epinephrine, and serotonin (Figure 1) [2], [3], [4]. These disorders are caused by a defect in the metabolism of tetrahydrobiopterin (BH4), the co-factor for phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH), or a defect at the level of TH or aromatic amino-acid decarboxylase (AADC) (Figure 1) [2], [3], [4]. Each of these conditions thus exhibits a characteristic CSF profile of pterins and of the precursors and metabolites of dopamine and serotonin (Table 1).

Figure 1:

Neurotransmitter metabolism.

Tyr and Trp Ohases, Tyrosine and tryptophan hydroxylases; DBH, dopamine β-hydroxylase; AADC, amino-acid decarboxylase; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 3-OMD, 3-ortho-methyl DOPA; 5-OHT, 5-hydroxytryptophan; MHPG, 3-methoxy-4-hydroxyphenylglycol.

Several enzymes are involved in BH4 synthesis from guanosine triphosphate (GTP) (Figure 2). Mutations in GTP cyclohydrolase (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) may lead to a defect in BH4 synthesis or regeneration (Table 1). Defects of BH4 are usually associated with hyperphenylalaninemia (HPA), but two BH4 deficiencies due to GTPCH-1 or SR defects, normally occur without HPA. The diagnosis of these conditions can be established through the analysis of neopterin (N) and biopterin (B) in CSF [2], [3], [4].

Figure 2:

Tetrahydrobiopterin (BH4) biosynthesis and regeneration (according to [1–4], and [9]).

GTP, Guanosine triphosphate; GTPCH, guanosine triphosphate cyclohydrolase; NH₂, dihydroneopterin; N, neopterin; PTPS, 6-pyruvoyl-tetrahydropterin synthase; SR, sepiapterin reductase; PCD, pterin-4α-carbinolamine dehydratase; DHPR, dihydropteridin reductase; BH₂, dihydrobiopterin; DHFR, dihydrofolate reductase; B, biopterin. Dashed arrows: non-enzymatic.

Neurotransmitter disorders caused by a deficiency in AADC or TH are characterized by a normal CSF pterin (P) pattern (Table 1). In the case of AADC deficiency, there is a strong reduction of CSF concentrations of homovanillic acid (HVA) and 5-hydroxy-indoleacetic acid (5-HIAA) with a concomitant increase of their precursors 3-ortho-methyl DOPA (3-OMD) and 5-hydorxy-tryptophan (5-OHT). As AADC is a pyridoxal 5′-phosphate dependent enzyme, the same CSF profile may be also observed in the case of vitamin B6 deficits or in the case of pyridox(am)ine-5′-phosphate oxidase (PNPO) deficiency. In the case of TH deficiency there is a reduced CSF concentration of HVA and/or a reduced HVA/5-HIAA ratio [2], [3], [4].

Some cases of an isolated decrease of 5-HIAA were also reported but without proof of TPH inherited disorder [2], [3], [4]. More recently, a dopamine transport deficiency syndrome has been described. The involved mechanism affecting the dopamine transport to presynaptic neurons is linked to a defective reuptake leading to dopamine accumulation in the synapse as reflected by the increase of HVA concentration and HVA/5-HIAA ratio in CSF [5].

Early diagnosis of neurotransmitter disorders is of importance notably because some of these diseases can be well treated [6]. The correct diagnosis of these conditions can be achieved through the analysis of HVA, 5-HIAA, 3-OMD, 5-OHT, neopterin, and B in the CSF (Table 1). However, while the analysis of neurotransmitter metabolites can be easily achieved by liquid chromatography coupled to electrochemical detection (ECD) [7] or to mass spectrometry (MS) [8] most of available analytical methods of pterins in CSF cannot allow direct quantification of BH4 [9].

This review will focus on the methods and advances for monitoring pterins in CSF. Current analytical methods are mostly based on earlier BH4 oxidation investigations [10], [11], [12], [13]. They have in common at least one chromatographic separation step. But they differ in the used detection mode and their ability to directly analyze native BH4. Subsequently, LC-MS/MS techniques have been developed that have sufficient sensitivity to quantitate the various pterin species that have been identified in CSF.

After a brief description of the earlier studies on the oxidation of BH4, we will further review the current methods of pterins determination in CSF.

Earlier studies on BH4 oxidation

Since the elucidation of its pivotal role in the hydroxylation reactions of living organisms [10], several studies using liquid chromatography coupled to UV-visible spectroscopy or to fluorescence detection (FD) [11], [12], [13], have been devoted to the elucidation of the mechanisms of oxidation of BH4.

Earlier studies concluded that a large portion of B in tissues and fluids appears to be present in labile reduced forms [11]. As B in its fully oxidized form is stable and has strong fluorescent properties, a selective preliminary oxidation of the biological sample has been considered for pterins analysis. Since, the effects of iodine oxidation on reduced forms of B have been further analyzed by using high performance liquid chromatography (HPLC) [11]. One year later, the electrochemical behavior of BH4 has been extensively studied on pyrolytic graphite and it has been found that oxidation and reduction consisted of two-electron processes [14]. This led to the development of techniques that use ECD for the analysis of reduced pterins in biological materials [15], [16].

Based on the data of these earlier investigations, several chromatographic methods coupled to FD or ECD have been proposed for pterins analysis in CSF. The chromatographic behavior of pterins present in CSF is well known. Their separation can be easily achieved by reversed phase or HILIC modes (Figure 3) [8], [9], [12], [15], [16], [17].

Figure 3:

Pterins chromatographic profile as a function of the separation mode.

(A) Atlantis dC18; mobile phase: pH 7.4, 0.05 M sodium citrate/methanol (97/3, v/v). (B) Zic-HILIC; mobile phase: pH 7.4, 0.2 M ammonium formiate/acetonitrile (20/80, v/v). (A, B) Flow rate: 0.6 mL/min at 30°C. Detection 260 nm (Cf. [9] for more details).

Current HPLC methods can be primarily classified as a function of the detection mode. Alternatively, as these methods differ in the sample preparation procedure and the targeted forms of pterins, they can be also classified as direct or indirect methods.

HPLC-ECD-FD methods

In 1985, sequential ECD and FD were proposed for the simultaneous quantification of BH4, BH2, and NH2 in CSF [15]. Using a mobile phase with a pH adjusted to 5.2 in a reverse phase mode, BH4 is detected electrochemically in the redox mode, while BH2 and NH2 were detected by FD after post-column coulometric oxidation (PCCO-FD) into B and N, respectively. However, due to the large number of electroactive compounds in CSF, the specific detection of BH4 in the redox mode may be questioned. As ECD is less robust and less sensitive than FD for routine analyses, it may be wondered why the authors did not detect BH4 by FD after coulometric oxidation as they did for BH2 and NH2 [15].

Recently, the effects of pH on the separation and the electro-oxidation of the targeted pterins have been extensively studied [9]. The authors observed that high oxidation yields (≥50%) of BH2 and NH2 can be obtained irrespective of the pH range between pH 3 and pH 8. For BH4, however, a sufficient yield (about 50%) only occurs at pH higher than 7.4. At pH <6 the BH4 oxidation yield is about 20% and then it decreases to become negligible at pH equal to or less than 4. Hence the low oxidation yield of BH4 at pH <6.0 could explain why BH4 was not detected in the original publication using ECD followed by FD. This work allowed the authors to propose an HPLC method able to simultaneously analyze all forms of pterins including BH4, BH2, B and NH2 by FD after post-column coulometric oxidation (Figure 4) [9]. The results obtained in 99 patients who did not suffer from any known inborn error of metabolism showed that P concentrations in CSF vary from 7.6 to 76 nM (median, 32 nM), 3.1 to 17.6 nM (median, 9.5 nM), and 3.9 to 47.3 nM (median, 13.5 nM) for BH4, BH2, and NH2, respectively [9].

Figure 4:

Chromatographic profiles of (A) a normal CSF sample and (B) a CSF sample with elevated NH2 level, according to [9].

Obtained under the same conditions as for Figure 2A with fluorescence detection after post-column oxidation at +600 mV. Unknown peaks are unlabeled.

Parameters affecting the CSF concentration of pterins

As pterins are present in labile reduced forms in CSF, it is mandatory to control the technical and biochemical factors that can affect their concentrations, in order to ensure the robustness of the used analytical method and a correct diagnosis.

In order to prevent the auto-oxidation and the oxidation catalyzed by metal ions of BH4 before and during the chromatographic analysis, some authors proposed to add to the sample 1 mg/mL of dithioerythritol (DTE), a thiol reagent, and 1 mg/mL of diethylenetriaminepentaacetic acid (DETAPAC), a chelating agent [15], [35]. According to the authors, these additives protected BH4 for at least 5 h at 4°C and for at least 1 month at −70°C. In order to avoid any BH4 oxidation during the chromatographic step, the authors also propose to add 0.16 mM of DTE to the mobile phase [28].

Recently, the technical and biochemical factors that can affect P concentrations in the CSF have been extensively investigated [26]. According to the authors, N and B concentrations are not affected by the ventriculo-spinal gradient and pretreatment of CSF. Also, the pretreatment of the sample with trichloro-acetic-acid and DTE before oxidation with iodine results in the most accurate determination of N and B [36]. However, by using the methodology of iodine oxidation the authors only looked at total B and N and did not investigate the stability of BH4, BH2 or NH2. Under these conditions it is likely that the reduced pterins auto-oxidized to oxidized forms during sample handling.

More recently, the stability of the CSF samples at 10°C in the dark in the injector was checked after several hours [9]. According to the authors, they did not observe significant differences after 6 h. However, after 8 h, NH2 and BH4 concentrations decreased between 4% and 31% of their initial values. Thus, they concluded that under their conditions, samples should be analyzed not more than 6 h [9]. The authors also checked the stability of pterins in CSF samples frozen at −80°C immediately after collection. As compared with the initial values determined at day 7 after collection they did not observe significant differences after 24 months of storage. Hence, they avoided adding a preservative agent to the samples thus facilitating the collection procedure.

Conclusion

Current methods of pterins determination in CSF can be classified in two categories, those using an offline oxidation procedure of the reduced pterins and those using a post-column electrochemical oxidation. The former are indirect methods because they cannot allow the direct quantification of the reduced forms while the latter are direct methods as they do measure the concentrations of BH4, BH2, and NH2.

Although sufficient to establish the correct diagnosis of neurotransmitter disorders (Table 1), and although many remarkable advances in biomedical research have been made on basis of indirect measurements, the indirect methods of BH4 determination may present some drawbacks. They underestimate the reduced forms of pterins, they are very time consuming, and they are difficult to automate.

In contrast, direct methods are simple, rapid, and easy to automate. Besides, as they can measure BH4 concentrations they can be used for pharmacokinetic studies and for monitoring BH4 supplementations in patients with BH4 deficiency. Direct quantification of BH4 is also essential for studying the metabolism of this cofactor. To all these purposes, the recent LC-MS/MS method, which provides a more comprehensive evaluation of the pterins pathway, offers a significant improvement. At present the LC-MS/MS method should be considered as the only possible reference method.