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.

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.

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.

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.

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.

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.

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.

A long-term study of the excretion of folate and pterins in a human subject after ingestion of 14C folic acid, with observations on the effect of diphenylhydantoin administration.

After the administration of 2-14C folic acid to a human volunteer, urinary and fecal radioactivity, as well as urinary excretion of folate (Lactobacillus casei assay) and biopterin-like material (Crithidia fasciculata assay) were determined at intervals over a 129 day period of observation. From two 24 h urine samples erythroneopterin, bioterin, threoneopterin pterin, isoxanthopterin, and xanthopterin were isolated by chromatographic procedures, quantitated, and their specific activities were determined. The effect on the pattern of elimination of urinary radioactivity and biological activity resulting from the administration of diphenylhydantoin was studied on two occasions. Urinary radioactivity plots suggest the decay of two forms of folates with markedly different biological half lives. One short-lived (t 1/2 approximately 31.5 hr), corresponding to newly absorbed folate, and one long-lived (t 1/2 approximately 100 day) thought to represent the decay of body pools. Diphenylhydantoin does not alter the rate of elimination of the long-lived component but may accelerate losses of newly absorbed folate. The analysis of pterins does not support the hypothesis that diphenylhydantoin increases the breakdown of folates to pterins.

Defects of tetrahydrobiopterin synthesis and their possible relationship to a disorder of purine metabolism (the Lesch-Nyhan syndrome).

The metabolic pathways of pterin de novo synthesis, interconversion and salvage which lead to the tetrahydrobiopterin cofactor of phenylalanine 4-monooxygenase, tyrosine 2-monooxygenase and tryptophan 5-monooxygenase are reviewed and data on the enzymes which catalyze the individual steps are presented. Analogies drawn between the inborn errors of tetrahydrobiopterin production and the Lesch-Nyhan syndrome, in which purine salvage is deficient, are used as a basis for the hypothesis that the neurological manifestations of the Lesch-Nyhan syndrome are due to neurotransmitter imbalance which stems from an imbalance of the aromatic amino acid monooxygenase activities which are themselves due to impaired pterin biosynthesis. The latter arises because, in the absence of the hypoxanthine phosphoribosyltransferase catalyzed purine salvage pathway, the supply of GTP for the GTP cyclohydrolase reaction, which is the first reaction on the pterin de novo synthesis pathway, is reduced. It is proposed that the different aromatic amino acid monooxygenases are differentially affected by this constrained pterin production. The activities of those most directly related to the quantal production of the cerebral neurotransmitters dopamine, norepinephrine and 5-hydroxytryptamine are affected whereas liver phenylalanine 4-monooxygenase activity is not overtly impaired. The results of different lines of research which support this concept are cited, as is direct evidence for a selective reduction of dopamine production in the basal ganglia of patients with the Lesch-Nyhan syndrome. It is proposed that lack of GMP for functions, other than its role in pterin de novo synthesis, accounts for the features of the Lesch-Nyhan syndrome which do not occur when only tetrahydrobiopterin production is deficient as in the inborn errors of tetrahydrobiopterin synthesis.

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.

Quantitative determination of unconjugated pterins in urine by gas chromatography/mass fragmentography.

A gas chromatographic/mass fragmentographic method is described which permits the determination of unconjugated pterins in urine. After the addition of 6,7-dimethylpterin as an internal standard, the acidified urine samples are purified by liquid chromatography on Dowex-50 and Dowex-1 columns. The pterins are then converted to their corresponding trimethylsilyl derivatives and the base peaks of biopterin (m/e 409), neopterin (m/e 409) and 6,7-dimethylpterin (m/e 320) are determined. The method is sensitive and specific and permits the processing of large numbers of samples. By means of this method, the urinary excretion of biopterin and neopterin from 9 healthy subjects has been determined.

Altered urinary excretion of pteridines in neoplastic disease. Determination of biopterin, neopterin, xanthopterin, and pterin.

Urinary pteridine concentrations in healthy control subjects and patients with cancer and non-malignant diseases were determined by HPLC and TLC after partial purification by ion exchange and Sephadex chromatography. Elevated concentrations of neopterin were found in 70% of the 50 cancer patients investigated. In patients with non-Hodgkin's lymphoma (seven cases) or with liver metastases (12 cases) neopterin concentrations were significantly higher than in control subjects (p < 0.01). Biopterin was less frequently increased (22%). Xanthopterin was generally raised when neopterin and/or biopterin excretion was high. Neopterin/biopterin ratios were higher in some patients with cancer or with severe renal insufficiency than in controls. These findings suggest that alterations in pteridine metabolism are common in malignant disease. The pathogenic, diagnostic and therapeutic significance of these changes remains to be established.

Dihydroxanthopterinuria in phenylketonuria and lethal hyperphenylalaninemia patients.

7,8-Dihydroxanthopterin in identified in urine from phenylketonuria and lethal hyperphenylalaninemia patients. Because 7,8-dihyroxanthopterin is readily oxidised to xanthopterin, most of the characterisation was performed on xanthopterin. This finding indicates a gross disturbance of tetrahydrobiopterin metabolism in these patients. 7,8-dihydroxanthopterin or a related pterin may play a role in the pathogenesis of neurological symptoms in phenylketonuria and lethal hyperphenylalaninemia.

Diagnosis of variants of hyperphenylalaninemia by determination of pterins in urine.

Assessment of urinary pterins is proposed as a rapid method for recognition of the variants of hyperphenylalaninemia. This is achieved by means of oxidation of pterins by iodine in acidic and alkaline solutions and then by high performance liquid chromatography on a cation-exchange column with fluorimetric detection. In biopterin-synthetase deficiency, only neopterin accumulated; in dihydropteridine-reductase (DHPR) deficiency and in phenylketonuria, high levels of pterins are found, but BH4 levels, absent in the former and high in the latter, allow a differential diagnosis. Phenylalanine loads in the controls also lead to increased elimination of pterins, but with a pattern different from that found in phenylketonuria. This method can be used before dietary treatment and thus can be proposed for all newly detected hyperphenylalaninemic babies.

Urinary excretion levels of unconjugated pterins in cancer patients and normal individuals.

Urinary excretion levels of seven unconjugated pterins in healthy individuals and in cancer patients, most of whom were undergoing chemotherapy, were measured utilizing a newly developed high-pressure liquid chromatographic system. Excretion of pterins in the control group appears to be under strict metabolic control as the values obtained were confined within a small range. When the mean excretion levels in control subjects were compared with those in cancer patients, we found a significant increase in the excretion of xanthopterin, neopterin and pterin and a significant decrease in isoxanthopterin by cancer patients. Biopterin levels, on the other hand, were found only slightly but not significantly increased, whereas pterin-6-carboxylic acid and 6-hydroxymethylpterin were found to be excreted in approximately equal amounts in both groups. Urinary excretion levels of pterins were monitored for a period of nine months in a patient being treated with chemotherapy for metastatic ovarian carcinomatosis. We found that the excretion pattern of pterins appeared to correlate with the clinical status of the patient. These results indicate that a definite imbalance in pterin, and possibly folate metabolism, is associated with the presence of malignant diseases.

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.

Adsorptive stripping voltammetric analysis of some pterines in human urine.

The combined content of biopterin and he separate folic acid content of human urine have been quantitated by phase-selective alternating-current stripping voltammetry at the static mercury drop electrode. The use of disposable C18 cartridge allowed the samples to be separated into two fractions, one containing biopterin and neopterin and the other folic acid. The average concentration of biopterin and neopterin together was 704 ng ml-1; precision, 6.6% (RSD, n=7); mean recovery, 96%. The average concentration of folic acid was 836 ng ml-1; precision, 8.2% (RSD, n=8); mean recovery, 91%.

Urinary excretion of pterins in tumor-bearing patients.

In a group of 14 individuals without tumor disease and 125 patients with verified malignant tumors we checked the applicability of determination of the pterins concentration in urine by the procedure described by RAO et al. [3]. In total of 242 examinations were performed. The results were compared with the thin-layer chromatography and on the basis of these comparisons the method was modified. Correlation of the clinical conclusion and the values of tests was obtained in 76% of cases. In patients with undoubtedly persisting tumors the correlation was found in 73%, in a group of patients with the leukemia and lymphomas in 97%, with mammary carcinoma in 81%. Evaluation made in these patients prior to the chemotherapy initiation correlated in 100%. This simple test offers a possibility of the use in differential diagnosis of malignant process or in the course of the screening.

Tetrahydrobiopterin and inherited hyperphenylalaninemias.

Tetrahydrobiopterin deficiency, a variant of hyperphenylalaninemia, may be caused by deficiency of one of the following enzymes: guanosine triphosphate cyclohydrolase 1,6-pyruvoyltetrahydropterin synthase, dihydropteridin reductase and pterin-4a-carbinolamine dehydratase. The first two enzymes are involved in the biosynthesis of tetrahydrobiopterin, the last two in its regeneration. Although these diseases are rare, early detection by selective screening is essential for the treatment and outcome. Tetrahydrobiopterin deficiencies are very heterogenous ranging from mild forms requiring only marginal if any treatment to severe forms which are in some cases very difficult to treat. All variants of tetrahydrobiopterin deficiency can be differentiated from the classical phenylketonuria (PKU) by measurement of pterin metabolites in patients' urine, tetrahydrobiopterin loading test, and by dihydropteridine reductase activity in erythrocytes from the Guthrie card.