Improved LC-MS/MS method for the determination of 42 neurologically and metabolically important molecules in urine.

Simultaneous determination of kynurenines, neurotransmitters, pterins and steroids linked to various neurological and metabolic diseases have important diagnostic significance for related pathology and drug monitoring. An improved, sensitive and selective ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometric (UHPLC-MS/MS) method, based on our earlier publication, has been proposed for the quantitative measurement of 42 metabolites in human urine. The assay covers a larger number of analytes, uses an advanced, Waters Atlantis T3 chromatographic column and similarly meets the guideline of European Medicines Agency (EMA) on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all > 0.998. Intra-day and inter-day accuracy and precision were 87-118%, 81-120% and 2-20%, respectively including the lower limit of quantification (LLOQ). The assay is expected to facilitate the diagnosis and allows drug level monitoring from urine.

Sepiapterin Reductase Inhibition Leading to Selective Reduction of Inflammatory Joint Pain in Mice and Increased Urinary Sepiapterin Levels in Humans and Mice.

OBJECTIVE: To evaluate the antiinflammatory and analgesic effects of sepiapterin reductase (SPR) inhibition in a mouse model of inflammatory joint disease, and to determine whether urinary sepiapterin levels, as measured in mice and healthy human volunteers, could be useful as a noninvasive, translational biomarker of SPR inhibition/target engagement. METHODS: The collagen antibody-induced arthritis (CAIA) model was used to induce joint inflammation in mice. The effects of pharmacologic inhibition of SPR on thresholds of heat-, cold-, and mechanical-evoked pain sensitivity and on signs of inflammation were tested in mice with CAIA. In addition, mice and healthy human volunteers were treated with SPR inhibitors, and changes in urinary sepiapterin levels were analyzed by high-performance liquid chromatography. RESULTS: CAIA in mice was characterized by 2 phases: in the acute inflammation (early) phase, joint inflammation and heat-, mechanical-, and cold-induced pain hypersensitivity were present, while in the postinflammation (late) phase, no joint inflammation was observed but heat- and mechanical-induced hypersensitivity, but not cold hypersensitivity, were present. Inhibition of SPR in mice with CAIA significantly attenuated the heat-induced hyperalgesia in both phases, and the mechanical allodynia in the late phase. Signs of inflammation were unaffected by SPR inhibition. Urinary tetrahydrobiopterin levels, as a marker of inflammatory pain, were increased during inflammation in mice with CAIA (2-fold increase over controls; P < 0.05) and significantly reduced by SPR inhibition (P < 0.05 versus vehicle-treated mice). Increased urinary sepiapterin levels in the presence of SPR inhibition in both mice and healthy human volunteers were associated with high sensitivity (70-85%) and high specificity (82-88%) for the prediction of SPR inhibition/target engagement. CONCLUSION: SPR inhibition reduces the pain associated with joint inflammation, thus showing its potential utility as an analgesic strategy for inflammatory joint pain. In addition, SPR inhibition increases urinary sepiapterin levels, indicating the potential of this measurement as a noninvasive biomarker of target engagement of SPR inhibitors, such as sulfasalazine, a disease-modifying antirheumatic drug that is currently used as a first-line treatment for rheumatoid arthritis.

Pterins as diagnostic markers of exercise-induced stress: a systematic review.

OBJECTIVES: To evaluate pterins as diagnostic biomarkers of exercise-induced stress. DESIGN: Systematic review of the literature. METHODS: MEDLINE, Scopus and Web of Science were searched in March 2019 for relevant literature. We only considered in vivo studies of healthy humans that reported measurement of a pterin(s) in response to exercise or sport with no underlying prior disease or complication. Relevant articles were independently reviewed and resolved by consensus. RESULTS: We included 29 studies with 644 participants. We classified articles by running/hiking, cycling, rugby, mixed martial arts (MMA) or other. Eighty-six percent of studies measured a significant increase in a pterin in response to exercise. Changes in pterin concentrations were within 24h of the exercise-stimulus in 79% of studies and 17% measured a change from baseline greater than 48h post-exercise (49% did not measure or report beyond 48h). Neopterin or total neopterin (neopterin+7,8-dihydroneopterin) were the primary pterin measured (28 studies) and they were equally sensitive to exercise regardless of whether the stimulus was running, cycling, rugby, MMA or other. CONCLUSIONS: Neopterin and total neopterin increase in response to exercise-induced stress. Pterins may have limited capacity for monitoring long-term stress beyond 48h but further research is required.

Determination of Urinary Pterins by Capillary Electrophoresis Coupled with LED-Induced Fluorescence Detector.

Urinary pterins have been found as potential biomarkers in many pathophysiological conditions including inflammation, viral infections, and cancer. However, pterins determination in biological samples is difficult due to their degradation under exposure to air, light, and heat. Besides, they occur at shallow concentration levels, and thus, standard UV detectors cannot be used without additional sample preconcentration. On the other hand, ultra-sensitive laser-induced fluorescence (LIF) detection can be used since pterins exhibit native fluorescence. The main factor that limits an everyday use of LIF detectors is its high price. Here, an alternative detector, i.e., light-emitted diode induced fluorescence (LEDIF) detector, was evaluated for the determination of pterins in urine samples after capillary electrophoresis (CE) separation. An optimized method was validated in terms of linearity range, limit of detection (LOD), limit of quantification (LOQ), intra- and interday precision and accuracy, sample stability in the autosampler, and sample stability during the freezing/thawing cycle. The obtained LOD (0.1 µM) and LOQ (0.3 µM) values were three-order of magnitude lower compared to UV detector, and two orders of magnitude higher compared to previously reported house-built LIF detector. The applicability of the validated method was demonstrated in the analysis of urine samples from healthy individuals and cancer patients.

The metabolic profiles of pterin compounds as potential biomarkers of bladder cancer-Integration of analytical-based approach with biostatistical methodology.

Cancer disease is the second leading cause of death across the world. The analysis of potential biomarkers of cancer can be useful in cancer screening or cancer diagnosis, and may provide valuable information on the disease risk and progression. Pterin compounds have been studied as candidates of potential biomarkers as their elevated levels have been reported in various cancer diseases. The objective of the study was to compare the profiles of six pterin compounds in urine of 35 healthy subjects and 46 patients diagnosed of bladder cancer with the use of HPLC coupled with fluorimetric detection. The results of the chromatographic analysis together with biostatistical-based approach showed, that the concentrations of pterin compounds in bladder cancer patients were higher as compared to healthy individuals, and statistically significant differences between patients and controls were reported for xanthopterin and isoxanthopterin. Moreover, gender-specific analysis revealed, that the concentrations of pterins in the group of women reached higher values in comparison to men. For metabolites juxtaposed in pairs, namely xanthopterin and isoxanthopterin as well as for neopterin and biopterin, we found significant positive correlations in the group of both, patients and healthy individuals. We therefore conclude, that chromatographic analysis with simultaneous extensive biostatistical-based interpretation of the metabolite profiles may provide deeper understanding of the relationships between pterin metabolites. The results do not prejudge the possibility of using pterin compounds in the diagnosis of bladder tumors. However the results may have an impact on the study of bladder cancer biomarkers.

Impact-induced muscle damage and urinary pterins in professional rugby: 7,8-dihydroneopterin oxidation by myoglobin.

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performance liquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10 µM and 50 µg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P < 0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress.

Urine sepiapterin excretion as a new diagnostic marker for sepiapterin reductase deficiency.

Sepiapterin reductase deficiency (SRD) causes depletion of biogenic amines in the brain, early onset motor disorder, and intellectual disability. The diagnostic marker for this rare disease is increased sepiapterin and biopterin in CSF. Through a new analytic methodology we demonstrated accumulation of sepiapterin in urine of four SRD patients several times greater than that found in healthy controls and carriers, regardless of age or treatment. Our findings suggest a new interpretation of current theories of peripheral pterin metabolism and provide a new noninvasive diagnostic tool for children with early onset cryptogenetic developmental delay and/or movement disorder.

Determination of pterins in urine by HPLC with UV and fluorescent detection using different types of chromatographic stationary phases (HILIC, RP C8, RP C18).

Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterin-6-carboxylic acid. The effect of mobile phase composition, buffer type, pH and concentration on retention using HILIC, C8 and C18 RP stationary phases were examined. Separation of pterins on RP and HILIC stationary phase was performed and optimized. Eight pterins were successfully separated on HILIC Luna diol-bonded phases, Aquasil C18 RP column and LiChrospher C8 RP column. Determination and separation of the pterins from urine samples were performed on HILIC Luna and LiChrospher C8 RP columns which were chosen as the most appropriate ones. Finally, LiChrospher C8 RP column with fluorescence detection was selected for further validation of the method. The optimum chromatographic condition was mobile phase methanol (A)/phosphoric buffer pH 7, 10mM (B), isocratic elution 0-15min 5% A flow=0.5ml/min 15-17min. 5% A, flow=0.5-1ml/min the linearity (R(2)>0.997) and retention time repeatability (RSD%<1) were at satisfactory level. The precision of peak areas expressed as RSD in % was between 0.55 and 14. Pterins detection limits varied from 0.041ng/ml to 2.9ng/ml. Finally, HPLC method was used for the analysis of pterins in urine samples with two different oxidation procedures. Concentration levels of pterin compounds in bladder cancer patients and healthy subjects were compared.

Determination of six pterins in urine by LC-MS/MS.

BACKGROUND: The present work describes an analytical method for urinary pterins by LC-MS/MS, with emphasis on the separation of 6- and 7-positional isomers of bio- and neopterins. RESULTS: Urine sample preparation consisted of oxidation by MnO(2), filtration and direct dilution in the mobile phase. The method was validated in urine spiked at five concentration levels with true triplicates of each level. Separation of the pterins, including the positional isomers, was achieved by employing a LUNA amino column. Six pterins were quantified (pterin, isoxanthopterin, 6-biopterin, 7-biopterin, 6-neopterin, 7-neopterin) and a linear behavior was observed; LOD varied from 7 to 360 pg/ml and correlation coefficients above 0.98 were obtained for all pterins. In addition, pterin levels were evaluated in 41 urine samples of healthy subjects, in ten urine samples of patients with classical phenylketonuria (PKU) and in one with atypical PKU. CONCLUSION: The proposed method allowed to identify, separate and quantify six pterins in urine, using a simple and rapid sample preparation. The atypical PKU was unequivocally differentiated from the classical form, demonstrating that this method could be very useful for characterization and follow-up of diseases.

Two Greek siblings with sepiapterin reductase deficiency.

BACKGROUND: Sepiapterin reductase (SR) deficiency is a rare inherited disorder of neurotransmitter metabolism; less than 25 cases have been described in the literature so far. METHODS: We describe the clinical history and extensive cerebrospinal fluid (CSF) and urine examination of two Greek siblings with the diagnosis of SR deficiency. The diagnosis was confirmed by enzyme activity measurement in cultured fibroblasts and by mutation analysis. RESULTS: Both patients suffered from a progressive and complex L-dopa responsive movement disorder. Very low concentrations of the neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were observed in CSF. CSF neopterin and biopterin concentrations were abnormal in one case only, whereas in both cases sepiapterin concentrations were abnormally high and 5-hydroxytryptophan was undetectable. Urine concentrations of HVA, 5-HIAA and vanillyl mandelic acid (VMA) were decreased in both cases. Both patients had no detectable SR enzyme activity in primary dermal fibroblasts, and upon analysis of genomic DNA revealed the same homozygous point mutation introducing a premature stop codon into the reading frame of the SPR gene (mutant allele K251X). CONCLUSIONS: Our cases illustrate that, apart from HVA and 5-HIAA analysis, the specific quantification of sepiapterin in CSF, rather than neopterin and biopterin alone, is crucial to the final diagnosis of SR deficiency. In addition, urinary concentrations of neurotransmitter metabolites may be abnormal in SR deficiency and may provide an initial indication of SR deficiency before CSF analysis is performed. The known, impressive beneficial response of SR deficient patients to treatment with L-dopa, is illustrated again in our cases.

Detection of 28 neurotransmitters and related compounds in biological fluids by liquid chromatography/tandem mass spectrometry.

This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28 compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds used as internal standards. The identification of compounds is achieved using the retention times (RT) and the characteristic fragmentations of ionized compounds. The acquisition modes used for the detection of characteristic ions turned out to be complementary: the identification of expected compounds only is feasible by MRM while expected and unexpected compounds are detected by NL. In the first part of this work, the fragmentations characterizing each molecule of interest are described. These fragmentations are used in the second part for the detection by MRM and NL of selected compounds in mixture with and without biological fluids. Any preliminary extraction precedes the analysis of compounds in biological fluids.

Screening for tetrahydrobiopterin deficiencies using dried blood spots on filter paper.

Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Phenylketonuria and glycogen storage disease type III in sibs of one family.

Hyperphenylalaninemia result from a block in the conversion of phenylalanine into tyrosine due to a defect in either the enzyme phenylalanine hydroxylase (98% of subjects) or in the metabolism of the cofactor tetrahydrobiopterin. Phenylalanine hydroxylase deficiency is the most common form of inherited hyperphenylalaninemia disorders, with a prevalence between 1/4,000-1/40,000. Glycogen storage disease (GSD) type III is caused by debranching enzyme deficiency of glycogen degradation. The clinical features vary in relation to the localization of the enzyme defect. Two clinical entities exist: a combined hepatic myogenic form (GSD IIIa) and a purely hepatic form (GSD IIIb). The inheritance is autosomal recessive. We describe a Turkish family in which two girls were found to have phenylketonuria, while in two other sisters glycogen storage disease type III was diagnosed. The parents of these children are cousins and they have had 12 children.

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates.

Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 micromol/L) were investigated further to differentiate between phenylalanine hydroxylase (PAH) deficiency and variant hyperphenylalaninaemia (HPA) forms. In patients 1 and 2 a tetrahydrobiopterin (BH4) load caused a significant decrease of the plasma Phe levels. A combined phenylalanine/BH4 loading test was performed in patients 2, 3 and 4. In the latter two patients, plasma Phe concentrations completely normalized within 8 h after the BH4 load (20 mg/kg). Basal urinary pterins were normal in all four patients. The activity of dihydropteridine reductase (DHPR) was normal in patients 1, 2 and 3 and 50% of control values in patient 4 (not in the range of DHPR-deficient patients). In patient 3 a subsequent phenylalanine loading test with concomitant analysis of plasma biopterins revealed a normal increase of biopterin, excluding a BH4 biosynthesis defect. Pterins and neurotransmitter metabolites in CSF of patients 1, 3 and 4 were normal. DNA mutations detected in the PAH gene of patients 1-4 were A313T, and L367fsinsC; V190A and R243X; A300S and A403V; R241C and A403V. The results are suggestive for mutant PAH enzymes with decreased affinity for the cofactor BH4.

Molecular analysis and long-term follow-up of patients with different forms of 6-pyruvoyl-tetrahydropterin synthase deficiency.

UNLABELLED: The outcome of 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, the most common form of tetrahydrobiopterin (BH4) deficiency, depends on factors such as severity of the disease, type of mutation, time of diagnosis, and mode of treatment. We investigated five patients from four different families, four of them presenting with the severe form of PTPS deficiency and one with the mild peripheral form. In this study, missense (L26F, T67M, P87L, V124L, D136G, D136V) and nonsense (R15-16ins) mutations were detected by reverse transcriptase polymerase chain reaction and sequence analysis. Two patients with the severe form were compound heterozygotes (T67M/P87L and D136G/R15-16ins), two siblings were homozygous for the D136V mutation, and in the patient with the mild form, heterozygous L26F/V124L mutations were present. Two patients are on combined therapy with L-dopa/carbidopa/5-hydroxytryptophan plus BH4, the siblings are on monotherapy with BH4, and the patient with the mild form is now off treatment, presenting with normal plasma phenylalanine levels. CONCLUSION: Long-term follow-up shows that the outcome of 6-pyruvoyl-tetrahydropterin synthase deficiency benefits from treatment started in the first months of life and that the phenotype may change with age. Additionally, depending on the type of mutations, prenatal damage to the fetus may multiply the clinical abnormalities and thus worsen the prognosis of the disease. In patients initially diagnosed with the mild peripheral form of the disease, therapy with tetrahydrobiopterin should be stopped after some time to test whether hyperphenylalaninaemia was only a transient condition.

Tetrahydrobiopterin responsiveness in phenylketonuria differs between patients with the same genotype.

Recently, BH(4)-responsive phenylalanine hydroxylase (PAH) deficiency was reported in patients with specific mutations in the PAH gene, and it was suggested that BH(4) responsiveness may be determined by the respective genotypes. We now report on three patients with PAH deficiency and the same genotype but different responses to standardized BH(4) loading. Our results suggest that BH(4) responsiveness in PAH deficiency is at least partly independent from PAH genotype.

Abnormalities in urinary pterin levels in Rett syndrome.

The pterins, neopterin and biopterin, occur naturally in body fluids including urine. Increased neopterin levels are associated with activation of the cellular immune system and reduced biopterins are essential for biosynthesis of the monoamine neurotransmitters. The present study measured urinary neopterin and biopterin by high-performance liquid chromatography in 40 subjects with Rett syndrome, eight of their healthy sisters and 29 female control volunteers (age range 2-54 years). The results confirm earlier preliminary findings that urinary neopterin levels are raised in a proportion of young girls with Rett syndrome but not in the older women. In contrast urinary biopterin levels are not different from controls in the youngest children but remain low while control values increase with age. These findings may indicate immune activation during the regression phase of Rett syndrome but also raise the possibility that an inherited fault in tetrahydrobiopterin metabolism increases the risk of developing the disorder.

The role of pterins and related factors in the biology of early postpartum depression.

Plasma tryptophan and other putative amino acids, cortisol, folate and vitamin B12 and urinary biopterin (B) and neopterins (N) were measured in three groups of women: 62 women in the early postpartum period, 23 pregnant and 38 non-gravid controls. Sixty-two postpartum women were screened for depression by the Edinburgh postnatal depression scale (EPDS) on day 7 after delivery. Postpartum women had significantly lower tryptophan, vitamin B12 and significantly greater levels of cortisol, folate, neopterins and biopterins than controls. Comparisons between women who were classified on the EPDS as cases and non-cases revealed only a statistically significant difference for lower N:B (P<0.01) and lower folate (P<0.01) ratio in cases than non-cases. Multiple regression analysis showed a significant contribution for low tryptophan to increased EPDS which also showed significant correlations with low methionine, low tyrosine, low N:B ratio and high vitamin B12.

Mutations in the pterin-4alpha-carbinolamine dehydratase (PCBD) gene cause a benign form of hyperphenylalaninemia.

Four patients with primapterinuria, postulated to be due to pterin-4alpha-carbinolamine dehydratase (PCD) deficiency, were diagnosed by biochemical and DNA analysis. All four patients presented in the neonatal period with hyperphenylalaninemia, and elevated neopterin and decreased biopterin levels in the urine. These symptoms are common to 6-pyruvoyltetrahydropterin synthase deficiency and thus there is a danger of misdiagnosis. In addition, all four patients had elevated urinary excretion of primapterin (7-biopterin), the only persistent biochemical abnormality. Analysis of fibroblast DNA from the patients identified the following mutations in the PCBD gene: one patient homozygous for the missense mutation E96K and one homozygous for the nonsense mutation Q97X, both in exon 4; one compound heterozygote with the mutations E96K and Q97X; and one patient with two different homozygous mutations: E26X in exon 2 and R87Q in exon 4. In two families, the parents were investigated and found to be obligate heterozygotes for particular mutations. One sibling was found to be unaffected. These results further substantiate the idea that primapterinuria is associated with mutations in the PCBD gene.

Inhalation of the nitric oxide synthase cofactor tetrahydrobiopterin in healthy volunteers.

Pulmonary endothelial dysfunction is the hallmark of acute lung injury. Impaired pulmonary endothelial nitric oxide (NO) production in this event has been described. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase and modulator of its activity. At high local concentrations, BH4 provokes local vasodilation in vivo in healthy individuals. At lower concentrations, BH4 selectively and locally restores disturbed NO-dependent vasodilation in patients with endothelial dysfunction. In this preliminary study, we therefore investigated the feasibility of BH4 inhalation in five healthy human volunteers. Inhalation of buffered, aqueous BH4-dihydrochloride solution was well tolerated; despite the buffer, BH4 stability was completely preserved. Resorption of inhaled BH4 was demonstrated by significantly increased BH4 levels in plasma and urine. Inhaled BH4 did not alter pulmonary function and had no effect on systemic hemodynamic values. Our data demonstrate that inhalation is a novel method for local BH4 administration, offering a basic therapeutic tool for investigation of restoration of impaired NO-dependent vasodilation due to pulmonary endothelial dysfunction.