Urinary neopterin and biopterin indicate that inflammation has a role in autism spectrum disorder.

Inflammation is thought to be involved in the pathogenesis of autism spectrum disorder (ASD). Pteridine metabolites are biomarkers of inflammation that increase on immune system activation. In this study, we investigated the urinary pteridine metabolites in ASD patients as a possible biomarker for immune activation and inflammation. This observational, cross-sectional, prospective study collected urine samples from 212 patients with ASD and 68 age- and sex-matched healthy individuals. Urine neopterin (NE) and biopterin (BIO) levels were measured. Patients who had chronic disorders, active infection at the time of sampling, or high C-reactive protein levels were excluded. The urine NE and BIO concentrations were determined by high-performance liquid chromatography. The ratios of both NE and BIO to creatinine (CRE) were used to standardise the measurements. The NE/CRE and NE/BIO levels were significantly higher in ASD patients than controls. Univariate and multivariate models revealed a significant increase in NE/CRE and NE/BIO in ASD patients. There was a significant relationship between the NE/BIO [average area under the curve (AUC) = 0.717; range: 0.637-0.797] and NE/CRE (average AUC = 0.756; range: 0.684-0.828) ratios, which distinguished individuals with ASD from controls. The elevated NE/CRE and NE/BIO ratios suggest that inflammation and T cell-mediated immunity are involved in the pathophysiology of autism. NE/BIO could serve as a diagnostic inflammatory marker in the pathogenesis of ASD.

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model.

INTRODUCTION: Pteridines include folate-derived metabolites that have been putatively associated with certain cancers in clinical studies. However, their biological significance in cancer metabolism and role in cancer development and progression remains poorly understood. OBJECTIVES: The purpose of this study was to examine the effects of tumorigenicity on pteridine metabolism by studying a panel of 15 pteridine derivatives using a progressive breast cancer cell line model with and without folic acid dosing. METHODS: The MCF10A progressive breast cancer model, including sequentially derived MCF10A (benign), MCF10AT (premalignant), and MCF10CA1a (malignant) cell lines were dosed with 0, 100, and 250 mg/L folic acid. Pteridines were analyzed in both intracellular and extracellular contexts using an improved high-performance liquid chromatography-tandem mass spectrometry method. RESULTS: Pteridines were located predominately in the extracellular media. Folic acid dosing increased extracellular levels of pterin, 6-hydroxylumazine, xanthopterin, 6-hydroxymethylpterin, and 6-carboxypterin in a dose-dependent manner. In particular, pterin and 6-hydroxylumazine levels were positively correlated with tumorigenicity upon folate dosing. CONCLUSIONS: Folic acid is a primary driver for pteridine metabolism in human breast cell. Higher folate levels contribute to increased formation and excretion of pteridine derivatives to the extracellular media. In breast cancer, this metabolic pathway becomes dysregulated, resulting in the excretion of certain pteridine derivatives and providing in vitro evidence for the observation of elevated pteridines in the urine of breast cancer patients. Finally, this study reports a novel use of the MCF10A progressive breast cancer model for metabolomics applications that may readily be applied to other metabolites of interest.

Synchronous luminescence spectroscopic characterization of urine of normal subjects and cancer patients.

Urine is one of the diagnostically potential bio fluids, as it contains many metabolites and some of them are native fluorophores. These fluorophores distribution and the physiochemical properties may vary during any metabolic change or at different pathologic conditions. Since urine is a multicomponent fluid, synchronous luminescence technique, a powerful tool has been adopted to analyse multicomponents in single spectrum and to resolve emission spectrum without much of photobleaching of fluorophores. In this study, urine samples of both normal subjects and cancer patients were characterised using synchronous luminescence spectroscopy with a Stokes shift of 20 nm. Different ratio parameters were calculated from the intensity values of the synchronous luminescence spectra and they were used as input variables for a multiple linear discriminant analysis across normal and cancer groups. The stepwise linear discriminant analysis classifies 90.3% of the original grouped cases and 88.6% of the cross-validated grouped cases correctly.

Simultaneous detection of six urinary pteridines and creatinine by high-performance liquid chromatography-tandem mass spectrometry for clinical breast cancer detection.

Recent preliminary studies have implicated urinary pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary pteridines: 6-biopterin, 6-hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the pteridines is between 0.025 and 0.5 µg/L, and for creatinine, it is 0.15 µg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary pteridines as breast cancer biomarkers.

Fluorescence characteristics of human urine from normal individuals and ovarian cancer patients.

Early diagnostics of ovarian cancer is difficult, because there are no symptoms until the disease has progressed to an advanced stage. As urine contains many intrinsic fluorophores, modern fluorescence techniques are perspective candidates for new routine urine tests. The presented work deals with differences in the fluorescence of metabolites in urine of ovarian cancer patients comparing to healthy volunteers using the fluorescence excitation-emission matrices. The most serious differences were found in undiluted urine at the fluorescence emission wavelengths from 400 nm to 460 nm when excited at 310 - 390 nm. Statistical analyses of our data have shown a 5-fold reduction in the intensity of the peak at 330/420 nm (excitation/emission wavelength) for undiluted urine samples excreted by cancer patients as compared to those of normal donors. Moreover, the ratio of intensities of the peaks at 370/440 nm and at 330/420 nm is 18-times elevated in urine excreted by patients with ovarian cancer as compared to healthy urine samples. The observed changes could be interpreted as reduction of the presence of pyridoxic acid, whereas blue-fluorescing pteridines becomes dominant in excitation-emission matrices of cancer urine samples in comparison to healthy donors. We suggest pteridines, which are related to cellular metabolism, as suitable candidates for neoplasia-associated fluorescent markers in human urine. Our work showed that monitoring of human urine fluorescent metabolites offers an alternative for ovarian cancer screening.

Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS.

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5-10.5 ng mL(-1) for neopterin, biopterin, and pterin; 4.0-8.0 ng mL(-1) for xanthopterin; and 0.5-4.5 ng mL(-1) for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min(-1), and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.

Separation and determination of 11 marker pteridines in human urine by liquid chromatography and fluorimetric detection.

A simple liquid chromatographic method has been developed to achieve the complete separation and determination of a wide range of pteridinic compounds and creatinine (CREA) in urine samples, in just one run. The influences of mobile phase composition and buffer pH have been studied. The optimized mobile phase was composed of a Tris-HCl buffer (15 mmol/L) at pH 6.10 solution (eluent A) and a Tris-HCl buffer (15 mmol/L) at pH 6.40 solution (eluent B), in gradient mode. Analytes were determined by fluorimetric detection, exciting at 272 nm, and measuring the fluorescence emission at three wavelengths, 410, 445 and 465 nm. CREA, as a reference of metabolites excretion in urine, was determined by photometric detection at 230 nm. Pteridines detection limits varied from 0.2 to 6.1 ng/mL, and 0.2 g/mL for CREA. Calculated precision values expressed as RSD (%) varied from 1.1 to 5.9. Two different oxidation procedures for urine samples were optimized. The neopterin/biopterin ratios found were 0.98 and 0.86 for adults and children, respectively, by means of the alkaline iodide/iodine oxidation and 0.45 and 0.57 using neutral KMnO(4) oxidation.

Lessons from 30 years of selective screening for tetrahydrobiopterin deficiency.

In addition to being hyperphenylalaninemic, patients lacking tetrahydrobiopterin (BH4) are deficient in the neurotransmitters whose synthesis depends on the normal activity of tetrahydrobiopterin-dependent tyrosine and tryptophan hydroxylases. Consequently, these patients have to be rapidly recognized among hyperphenylalaninemic babies, since they need specific and early substitutive therapy. Since 1980, BH4 metabolism has been investigated in 2,186 hyperphenylalaninemic babies, using HPLC measurement of pteridines in urine to recognize tetrahydrobiopterin synthesis deficiency (GTP cyclohydrolase and PTPS deficiency) and direct DHPR assay in dried blood samples to recognize DHPR deficiency. A total of 73 tetrahydrobiopterin deficient patients have been detected. Considering the group of neonates born in France (1,342), out of the 32 BH4 deficient patients which have been detected, only 8 were from caucasian families. The lessons from that experience are: (1) tests on blood and urine collected on filter paper cards commend itself by their convenience and simplicity, and samples can be collected on the first visit of the screened infants to the out-patient clinic; and (2) the preconceaved idea that newborns with moderate elevation of blood phenylalanine are false positives of the screening or mild forms of hyperphenylalaninemia explains that a significant number of cases were investigated after 1 month of age; however, in half of BH4-deficient babies, blood phenylalanine was below 10 mg/dl (0.6 mmol/l).

A simple and useful method for evaluation of oxidative stress in vivo by spectrofluorometric estimation of urinary pteridines.

Pteridine derivatives are intermediate metabolites of folic acid and its cofactors. Oxidized-form pteridines, but not reduced-form pteridines, are fluorescent substances. The purpose of this study was to clarify whether oxidized-form pteridine level in urine, estimated by spectrofluorometry, reflects oxidative stress in vivo. The subjects were healthy middle-aged men (n = 258). Urinary pteridine level was estimated by spectrofluorometry with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Relationships of urinary pteridines with oxidative stress markers (urinary DNA/RNA oxidation products and 15-isoprostane F2t) and with smoking were analyzed. Concentrations of pteridines, DNA/RNA oxidation products and 15-isoprostane F2t were used after logarithmic transformation in linear analyses. Pteridine levels were significantly correlated with levels of DNA/RNA oxidation products (Pearson's correlation coefficient: 0.626, p < 0.01) and 15-isoprostane F2t (Pearson's correlation coefficient: 0.695, p < 0.01). These correlations were not confounded by age, body mass index, history of smoking and estimated glomerular filtration rate in multivariate analysis. The mean urinary pteridine level was significantly higher in heavy smokers (16 cigarettes or more per day) than in nonsmokers and light smokers (less than 16 cigarettes per day) and was higher in light smokers than in nonsmokers. Thus, urinary fluorometric pteridine levels were shown to be associated with known biomarkers of oxidative stress as well as smoking, which causes oxidative stress in vivo. We propose spectrofluorometrical estimation of urinary pteridines as a simple and useful method for evaluation of oxidative stress in vivo.

Optimization of urinary pteridine analysis conditions by CE-LIF for clinical use in early cancer detection.

The second leading cause of death in the US is cancer and early discovery of the disease has translated into reduced fatality rates. We have identified and performed a systematic investigation of a method for urinary pteridine analysis by using CE-LIF, which is believed to possess the potential to diagnose the presence of cancer even earlier than existing methodologies. Through system enhancements, we have been able to improve the resolution of the two least resolved sets of peaks (6,7-dimethylpterin versus 6-biopterin and D-(+)-neopterin versus 6-hydroxymethylpterin) from 0.85 to 2.48 and 0.90 to 3.58, respectively. Additionally, we have discovered that the preparation of the urine samples in previous works was inadequate, and we have corrected the method to fully oxidize the pteridines in the urine, resulting in significantly less variability in quantification and greater ease of defining p-values for healthy versus cancer patients. Finally, we have performed validation steps of spike and recovery and short-term aging studies to demonstrate the method's robustness. As a result, we present an optimized and validated method ready for transfer from discovery phase to clinical trial that can potentially act as a non-invasively pre-screening test for cancer.

Molybdenum cofactor biosynthesis in humans. Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor.

Molybdenum cofactor deficiency is a devastating disease with affected patients displaying the symptoms of a combined deficiency of sulfite oxidase and xanthine dehydrogenase. Because of the extreme lability of the isolated, functional molybdenum cofactor, direct cofactor replacement therapy is not feasible, and a search for stable biosynthetic intermediates was undertaken. From studies of cocultured fibroblasts from affected individuals, two complementation groups were identified. Coculture of group A and group B cells, without heterokaryon formation, led to the appearance of active sulfite oxidase. Use of conditioned media indicated that a relatively stable, diffusible precursor produced by group B cells could be used to repair sulfite oxidase in group A recipient cells. Although the extremely low levels of precursor produced by group B cells preclude its direct characterization, studies with a heterologous, in vitro reconstitution system suggest that the precursor that accumulates in group B cells is the same as a molybdopterin precursor identified in the Neurospora crassa molybdopterin mutant nit-1, and that a converting enzyme is present in group A cells which catalyzes an activation reaction analogous to that of a converting enzyme identified in the Escherichia coli molybdopterin mutant ChlA1.

Evaluation of pteridine metabolism in battery workers chronically exposed to lead.

Occupationally-exposed lead affects the neuromuscular junction and might cause disturbances in the locomotor activity. This study was undertaken to evaluate pteridine metabolism, in which neurotransmitters are synthesized in battery workers. Urinary neopterin, biopterin and creatinine were measured using high performance liquid chromatography. Serum neopterin concentrations were detected by enzyme-linked immunoassay. Blood dihydropteridine reductase (DHPR) activities and deltaaminolevulinic acid (delta-ALA) were measured spectrophotometrically. Blood and urinary lead were detected by atomic absorption spectroscopy. Significantly increased blood and urinary lead levels, urinary neopterin, biopterin and delta-ALA were found in workers, while DHPR activities were indifferent compared to control group. Urinary creatinine decreased. This is the first study to demonstrate that increased activity of the pteridine pathway results in the accumulation of the neurotransmitters that may be responsible for the neurological disorders.

Development of a high-performance liquid chromatography - Tandem mass spectrometry urinary pterinomics workflow.

Pteridines have evoked considerable interest from the scientific community owing to their prominent roles in human health and disease. The availability of analytical methodologies suitable for comprehensive pteridine profiling, termed here as "pterinomics", has been limited by inconsistent sample preparation and the exclusion of lesser studied pteridine derivatives. In response, the present study describes a new pterinomics workflow using a high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS) methodology for the simultaneous analysis of 15 pteridine derivatives including four structural isomers, marking the largest quantitative pteridine panel that has been studied to-date. The validated method possessed excellent sensitivity with method detection limits (0.025 µg L(-1) to 0.5 µg L(-1)) that were comparable or superior to existing techniques. Spiked recovery studies demonstrated the technique was both accurate (88-112%) and precise (RSD: 0-6%). A comparative study of commonly used oxidative pretreatments, including triiodide, permanganate, and manganese dioxide, revealed that the oxidative mechanisms were inefficient, complex, and concentration dependent. Finally, 50 clinical urine specimens were examined with the new technique wherein 10 pteridine derivatives were quantified and population ranges have been given. This technique can be used to examine pteridine molecular epidemiology and biochemistry to support related research applications, and may further be readily extended to include additional pteridine derivatives and biological matrices for specific applications.

Chromatographic behavior of 12 polar pteridines in hydrophilic interaction chromatography using five different HILIC columns coupled with tandem mass spectrometry.

Retention characteristic of 5 hydrophilic interaction chromatography (HILIC) columns, containing neutral and possibly negatively charged support (silica, diol and amide), cationic phase (triazole) and zwitterionic phase (sulfobetaine), that are commercially available were studied for the separation of a group of 12 polar pteridines. The main factors influencing the retention and selectivity of pteridines for these different HILIC systems have been studied in liquid chromatography-tandem mass spectrometry (LC-MS/MS) conditions: mobile phase composition, buffer type, pH and concentration and the separation mechanism was also investigated. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange, and optimization of HILIC analyses depends on the ionization state of the analytes. For silica, diol, amide and sulfobetaine phases, hydrophilic partitioning mainly contributes to the retention, while electrostatic interactions and hydrogen-bonding should be considered to understand the elution orders for triazole phase. An zwitterionic phase (ZIC-HILIC) provided the stronger retention for all pteridines than other tested columns.

The turnover catabolism and excretion of folate administered at physiological concentrations in the rat.

This study examines the distribution of folate-derived compounds in rat urine on a daily basis after the administration of tracer doses of radioactive [3H]pteroylglutamic acid. The identification of 10-formyldihydropteroyl-glutamate in the rat urine, prior to equilibration of the tracer, is also reported for the first time.

Effect of ionizing radiation on the pteridine metabolic pathway and evaluation of its cytotoxicity in exposed hospital staff.

Investigations carried out to estimate the effect of long-term occupational exposure to low levels of external ionizing radiation indicated that exposed hospital staff showed an increase in chromosome aberrations. The purpose of this study was to evaluate whether genomic instability or an alteration in pteridine synthesis could be used as a marker of the potential hazard of ionizing radiation in hospital workers. Twenty gamma-radiation- and 33 X-ray-exposed technicians working in radiotherapy and radio-diagnostic units were included in this study, along with 22 healthy matched individuals. Plasma concentrations of nitrite plus nitrate (NO(x)) were measured to estimate reactive nitrogen species. Urinary neopterin, biopterin and creatinine concentrations were measured by high-performance liquid chromatography to determine metabolic activity along the pteridine pathway. Sister chromatid exchange was used as a measure of mutagenicity. Apoptosis was evaluated morphologically and also with a DNA-fragmentation test. The plasma NO(x) levels of both gamma-radiation- and X-ray-exposed technicians were significantly higher than those of the healthy controls (p<0.05). While the urinary biopterin concentrations were significantly higher in radiation-exposed groups compared with the healthy subjects (p<0.05), urinary neopterin concentrations remained unchanged. The apoptosis rates of gamma-radiation- and X-ray-exposed workers were significantly elevated in comparison with those in the control group (both p<0.05). Also, the increase in sister chromatid exchange frequency was significant in each of the radiation-exposed groups (exposed groups versus controls; p<0.05). These results indicate that long-term exposure to low-dose ionizing radiation, even below the permitted levels, could result in increased oxidative stress, which may lead to DNA damage and mutagenicity.

A rebuttal to "A comment to 'Normalization of urinary pteridines by urine specific gravity for early cancer detection' [Clin. Chim. Acta 435 (2014) 42-47]".

We really appreciate the comments from Drs. Reibnegger and Fuchs regarding our recent publication "Normalization of urinary pteridines by urine specific gravity for early cancer detection [Clin. Chim. Acta 435 (2014) 42-47]". In their letter, Drs. Reibnegger and Fuchs identify several potential concerns regarding our recent publication [1] that evaluated the normalization performance of urine specific gravity (USG) and urinary creatinine with respect to the diagnostic properties of selected pteridines in discerning aggressive and benign breast cancers. Their letter not only provides unique insights that are both relevant and helpful to many researchers engaging in similar studies, but also provides a wonderful opportunity for us to address these potential concerns that may also be shared by other readers. We addressed all of the comments by Drs. Reibnegger and Fuchs in this letter.

Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals.

Pteridines belong to a class of fluorescent metabolites that are excreted by humans in urine and their concentrations can reflect various pathophysiological states. We quantified the differences in urinary pteridine levels in patients with malignant and benign ovarian tumors and in healthy individuals. Urine samples were centrifuged and supernatants were oxidized by MnO2 before analysis. Levels of neopterin, biopterin, and pterin were assessed by fluorescence analysis of human urine after HPLC separation. We have revealed that the median neopterin levels were higher in urine samples from patients with malignant (0.226 µmol/mmol creatinine) and benign ovarian tumors (0.150 µmol/mmol creatinine) than in healthy subjects (0.056 µmol/mmol creatinine). The median neopterin levels of patients with malignant tumors were higher (1.5-times) than in patients with benign tumors. The median biopterin level in urine of patients with benign ovarian tumors (0.268 µmol/mmol creatinine) was found to be very close to the level in patients with malignant ovarian tumors (0.239 µmol/mmol creatinine), and both were higher than in healthy samples (0.096 µmol/mmol creatinine). The levels of urine pterin followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels.

Neopterin as a new biochemical marker for diagnosis of allograft rejection. Experience based upon evaluation of 100 consecutive cases.

The use of daily urinary neopterin evaluation to detect immunological complications has been tested in 96 consecutive cadaveric kidney recipients, three liver recipients, and one pancreas recipient. In 29 of these patients an immunologically uncomplicated posttransplant course was associated with stable or low neopterin levels, or both. In only 5% of daily determinations on these patients were increasing or high neopterin levels seen. On the other hand, major immunological complications, such as acute rejection episodes (38 cases), viral infections (17 cases), or both problems (8 cases), were preceded by increasing or high neopterin levels or both--on the average by one day. Withdrawal of cyclosporine was also found to be followed by increase of urinary neopterin levels. Neopterin evaluation enabled reliable and accurate prediction of immunological complications in 95% of patients with acute rejections and in 100% of patients with viral infections. It thus appears that daily assessment of urinary neopterin levels represents a useful tool for biochemical detection of immunological complications in allograft recipients.

Neopterin as a new biochemical marker in the clinical monitoring of bone marrow transplant recipients.

In previous reports we demonstrated that increased amounts of the pyrazinopyrimidine compound neopterin are released in the context of T lymphocyte activation. The aim of this investigation was twofold: (1) to define the contribution of hemopoetic cells to neopterin excretion, and (2) to search for the clinical utility of this biochemical marker in the monitoring of such patients. Thirteen patients were grafted with allogeneic, 1 with syngeneic, and 2 with autologous marrow. Urinary neopterin excretion was measured daily by means of high-performance liquid chromatography from the time before transplantation until the patients' discharge from the isolation unit. In all patients bone marrow aplasia was associated with depressed, and engraftment with increased, neopterin values. Rising neopterin levels invariably preceded the cytological definition of "take," on the average by seven days. After hematological reconstitution, neopterin excretion continuously declined in all 5 patients lacking infectious complications and/o-graft-versus-host disease (GVHD). A transitory increase of urinary neopterin followed by normalization was observed in 5 further patients. At the time of increased neopterin excretion, 4 experienced either herpetic infection or GVHD, both of which resolved promptly under the appropriate treatment. Neopterin values remained elevated after engraftment in 6 patients who suffered from persistent GVHD. Results of this pilot study suggest that (1) bone marrow derived cells are crucially involved in production of neopterin in vivo and (2) evaluation of neopterin excretion patterns after hemopoietic reconstitution enables one to discriminate between patients with and without an increased risk of developing GVHD or viral disease.