LC-MS/MS Analysis of Cerebrospinal Fluid Metabolites in the Pterin Biosynthetic Pathway

Abstract

The analysis of (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and neopterin in cerebrospinal fluid (CSF) is often used to identify defects in the pterin biosynthetic pathway affecting monoamine metabolism that can lead to pediatric neurotransmitter diseases. Low levels of BH4 and neopterin alone may not be sufficient to determine the defect, and further testing is often required. We have developed a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of BH4, 7,8-dihydrobiopterin (BH2), neopterin, and sepiapterin in CSF, which provides a more comprehensive evaluation of the pterin pathway. The method utilizes labeled stable isotopes as internal standards and allows for a fast 10-minute analysis by LC/MS/MS over a linear working range of 3 to 200 nmol/L. Total analytical imprecision is less than 14.4% for all pterin metabolites. Accuracy for BH4 and neopterin was determined by comparing data obtained by an alternative method using HPLC with EC and fluorescence detection. Excellent correlation was demonstrated for BH4 (r = 0.9646, 1/slope = 0.9397; n = 28; concentration range 3 to 63 nmol/L) and neopterin (r = 0.9919, 1/slope = 0.9539; n = 13; concentration range 5 to 240 nmol/L). CSF specimens from patients diagnosed with inborn errors of sepiapterin reductase (SR), 6-pyruvoyl-tetrahydropterin synthase (PTPS), dihydropteridine reductase (DHPR), and guanosine triphosphate cyclohydrolase (GTPCH) have been analyzed, and distinct pterin metabolite patterns were consistent with the initial diagnosis. This method differentiates patients with DHPR and SR deficiency from other pterin defects (GTPCH and PTPS) and will be useful for the diagnosis of specific defects in the pterin biosynthetic pathway.

Fig. 1

Pterin and monoamine metabolism. Abbreviations: GTP guanosine triphosphate, GTPCH GTP cyclohydrolase, PTPS 6-pyruvoyl-tetrahydropterin synthase, SR sepiapterin reductase, BH4 (6R,S)-5,6,7,8-tetrahydrobiopterin, BH2 7,8-dihydrobiopterin, DHPR dihydropteridine reductase, PCD pterin-4-α-carbinolamine dehydratase, CR carbonyl reductase, DHFR dihydrofolate reductase, qBH2 quinine-dihydrobiopterin, TH tyrosine hydroxylase, TPH tryptophan hydroxylase, PAH phenylalanine hydroxylase, AADC aromatic amino acid decarboxylase, L-DOPA L-3,4-dihydroxyphenylalanine, 5-HTP 5-hydroxytryptophan, 3-OMD 3-o-methyldopa, DA dopamine, 5-HT serotonin, HVA homovanillic acid, 5-HIAA 5-hydroxyindoleacetic acid

Fig. 2

Product ion spectra of each pterin and a representative chromatograph of each metabolite in CSF. (a, BH4 = 51 nM; b, BH2 = 39 nM; c, neopterin = 20 nM; and d, sepiapterin = 23 nM). Spectra were generated with + ESI-MS/MS by infusion (10 μL/min) of pure standards (1 μM/L each)

Fig. 3

Pearson correlation coefficients (95% CLs) for BH4 and neopterin (a and b) were r = 0.9646 (0.9240, 0.9837) and r = 0.9919 (0.972, 0.998)%, respectively. Deming regression analysis for BH4 (a) yielded an intercept (95% CLs) of 1.05 (−1.16, 1.65) nmol/L and a 1/slope of 0.9397; neopterin (b) yielded an intercept of (95% CLs) of −7.17 (−2.86, −11.48) nmol/L and a 1/slope of 0.9539. Bland–Altman proportional bias analyses (c and d) relative bias (95% CLs) for BH4 and neopterin were −1.6 (4.1, −7.4)% and −2.9 (18.8, −24.5)%, respectively. All data expressed as nmol/L