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Prediction and early detection of kidney damage induced by nonsteroidal anti-inflammatories (NSAIDs) would provide the best chances of maximizing the anti-inflammatory effects while minimizing the risk of kidney damage. Unfortunately, biomarkers for detecting NSAID-induced kidney damage in cats remain to be discovered. To identify potential urinary biomarkers for monitoring NSAID-based treatments, we applied an untargeted metabolomics approach to urine collected from cats treated repeatedly with meloxicam or saline for up to 17 days. Applying multivariate analysis, this study identified a panel of seven metabolites that discriminate meloxicam treated from saline treated cats. Combining artificial intelligence machine learning algorithms and an independent testing urinary metabolome data set from cats with meloxicam-induced kidney damage, a panel of metabolites was identified and validated. The panel of metabolites including tryptophan, tyrosine, taurine, threonic acid, pseudouridine, xylitol and lyxitol, successfully distinguish meloxicam-treated and saline-treated cats with up to 75-100% sensitivity and specificity. This panel of urinary metabolites may prove a useful and non-invasive diagnostic tool for monitoring potential NSAID induced kidney injury in feline patients and may act as the framework for identifying urine biomarkers of NSAID induced injury in other species., Competing Interests: The authors have declared that no competing interests exist.

Using a non-targeted metabolomics platform, we recently identified C-mannosyltryptophan and pseudouridine as non-traditional kidney function markers. The aims of this study were to obtain absolute concentrations of both metabolites in blood and urine from individuals with and without CKD to provide reference ranges and to assess their fractional excretions (FE), and to assess the agreement with their non-targeted counterparts. In individuals without/with CKD, mean plasma and urine concentrations for C-mannosyltryptophan were 0.26/0.72 µmol/L and 3.39/4.30 µmol/mmol creatinine, respectively. The respective concentrations for pseudouridine were 2.89/5.67 µmol/L and 39.7/33.9 µmol/mmol creatinine. Median (25 th , 75 th percentiles) FEs were 70.8% (65.6%, 77.8%) for C-mannosyltryptophan and 76.0% (68.6%, 82.4%) for pseudouridine, indicating partial net reabsorption. Association analyses validated reported associations between single metabolites and eGFR. Targeted measurements of both metabolites agreed well with the non-targeted measurements, especially in urine. Agreement for composite nephrological measures FE and urinary metabolite-to-creatinine ratio was lower, but could be improved by replacing non-targeted creatinine measurements with a standard clinical creatinine test. In summary, targeted quantification and additional characterization in relevant populations are necessary steps in the translation of non-traditional biomarkers in nephrology from non-targeted discovery to clinical application.

Pseudouridine (Ψ) is an important urinary cancer biomarker, especially in human colorectal cancer (CRC). Disclosed herein is the first Ψ molecularly imprinted polymer (Ψ-MIP) material obtained from tailor-engineered functional monomers. The resulting MIP imprint exhibits a remarkable imprinting factor greater than 70. It is successfully used for the selective recognition of Ψ in spiked human urine. This selective functionalized material opens the route to the development of inexpensive disposable chemosensors for noninvasive CRC diagnosis and prognosis., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Post-stroke depression (PSD) is the most common psychiatric complication in stroke survivors that has been associated with increased physical disability, distress, poor rehabilitation, and suicidal ideation. However, there are still no biomarkers available to support objective laboratory testing for this disorder. Here, a GC-MS-based urinary metabolomics approach was used to characterize the urinary metabolic profiling of PSD (stroke) subjects and non-PSD (health controls) subjects in order to identify and validate urinary metabolite biomarkers for PSD. Six metabolites, azelaic acid, glyceric acid, pseudouridine, 5-hydroxyhexanoic acid, tyrosine, and phenylalanine, were defined as biomarkers. A combined panel of these six urinary metabolites could effectively discriminate between PSD subjects and non-PSD subjects, achieving an area under the receiver-operating characteristic curve (AUC) of 0.961 in a training set (n = 72 PSD subjects and n = 146 non-PSD subjects). Moreover, this urinary biomarker panel was capable of discriminating blinded test samples (n = 58 PSD patients and n = 109 non-PSD subjects) with an AUC of 0.954. These findings suggest that a urine-based laboratory test using these biomarkers may be useful in the diagnosis of PSD.

Background and Objectives: Chronic obstructive pulmonary disease (COPD) is accompanied by muscle involvement. Pseudouridine, a catabolite of RNA, has been used in other conditions to assess muscle catabolism. We have examined the excretion of pseudouridine in patients with different stages of COPDs evolution., Subjects and Methods: We have defined four population groups: control group (without disease), chronic bronchitis group, emerging COPD group, and advanced COPD group. Pseudouridine was determined by high performance liquid chromatography,, Results: Pseudouridine extraction (pseudouridine/creatinine ratio) was (mean+19.9 (6.6) μmol/mmol in control group and was found to be very increased in all the patients with pulmonary condition: chronic bronchitis, 44.1 (60.75) μmol/mmol, 81.6 (56.8) μmol/mmol in emerging COPD group and 140.1 (68) μmol/mmol in advanced COPD for all the comparisons with normal subjects and among patients with lung disease). Age and gender did not affect pseudouridine excretion., Conclusions: The urinary excretion of pseudouridine is increased in chronic bronchitis and COPD and is related to disease stage. Its excretion is independent of age and gender., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Urinary excreted RNA and DNA catabolites are used as noninvasive markers for metabolic processes: 8-oxo-2'-deoxyguanosine (8-oxodG) potentially represents oxidative stress to DNA/deoxyribonucleotidetriphosphate pool, modified ribonucleoside pseudouridine (psi) originating mainly from degraded rRNA and tRNA reflects RNA turnover. Modified amino acid gamma-carboxyglutamic acid (Gla) stems from degraded proteins reflecting turnover of proteins. Aim of the present study was to investigate (44 healthy males, 3-18 y) how excretion rates of 8-oxodG, psi, and Gla are related to resting metabolic rate and energy intake. Excretion rates of 8-oxodG (pmol/kg/d), psi (micromol/kg/d), and Gla (micromol/kg/d) were significantly correlated with resting metabolic rate (kJ/kg/d): r = 0.108 (p = 0.029), 0.691 and 0.552 (p < 0.0001), respectively. Excretion rates of 8-oxodG, psi, and Gla were also significantly correlated with energy intake (kJ/kg/d): r = 0.108 (p = 0.036), 0.602 and 0.462 (p < 0.0001). 8-oxodG and Gla excretion was significantly correlated with psi excretion: r = 0.174 (p = 0.005) and 0.709 (p < 0.0001). These results indicate close relationships between whole-body RNA and protein degradation and metabolic rate. The relationship between 8-oxodG excretion and metabolic rate, however, is less strong suggesting that factors other than metabolic rate considerably affect the oxidative stress to DNA.

Background: Pulmonary rehabilitation can improve the functional capacity, but has a variable effect on the low fat-free mass (FFM) in patients with chronic obstructive pulmonary disease., Hypothesis: Pulmonary rehabilitation would not affect catabolic drives such as systemic inflammation and also protein breakdown., Methods: Patients (n = 40) were studied at the start of an 8-week in-patient pulmonary rehabilitation programme, at the end of the programme and 4 weeks later. FFM and functional capacity (quadriceps strength, handgrip strength and peak workload) were assessed. Pseudouridine (PSU) urinary excretion (cellular protein breakdown) and inflammatory status were determined. Healthy participants had a single baseline assessment (n = 18)., Results: PSU, (IL)-6 and soluble tumour necrosis factor (sTNF)alpha R75 were increased in patients compared with healthy participants, whereas FFM and functional capacity were reduced (all p < 0.01). PSU was inversely related to both FFM and skeletal muscle function. FFM and functional parameters increased with rehabilitation, but PSU and inflammatory status were unaffected. The gain in FFM was lost 4 weeks after the completion of rehabilitation (p < 0.01)., Conclusion: The anabolic effect of pulmonary rehabilitation improved FFM, but it did not reverse the increased protein breakdown or systemic inflammation. Thus, on cessation of pulmonary rehabilitation the FFM gains were lost owing to a loss of anabolic drive.

Background: Some patients with malignant lymphoma do not manifest superficial lymphadenopathy. In such cases, clinical parameters that indicate the number of tumor cells are important for the assessment of tumor growth and choice of proper treatment. We evaluated urinary pseudouridine (U-PU) as an indicator of the growth of malignant lymphoma by comparing its levels with serum concentrations of other clinical parameters in patients with various lymphomas at various stages., Methods: Urine was obtained from 67 patients with lymphoma. U-PU was assayed by recombinant Fab-based inhibition ELISA. Serum soluble IL2 receptor (sIL2R), serum deoxythymidine kinase (dTK), serum beta-2 microglobulin (beta2MG) and serum lactate dehydrogenase (LDH) were also assayed., Results: U-PU concentrations showed good correlations with serum concentrations of beta2MG, LDH, sIL2R and dTK. The level of U-PU was higher in stage IV than in stages I (P=0.023), II (P=0.006) and III (P=0.036)., Conclusion: U-PU concentration correlates with the clinical stage of lymphoma and is a useful tool to assess the growth of lymphoma.

Background: Cachexia is common in chronic obstructive pulmonary disease (COPD) and is thought to be linked to an enhanced systemic inflammatory response., Objective: We investigated differences in the systemic inflammatory profile and polymorphisms in related inflammatory genes in COPD patients., Design: A cross-sectional study was performed in 99 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stages II-IV), who were stratified by cachexia based on fat-free mass index (FFMI; in kg/m2: <16 for men and <15 for women) and compared with healthy control subjects (HCs). Body composition was determined by bioelectrical impedance analysis. Plasma concentrations and gene polymorphisms of interleukin 1beta (IL-1beta -511), IL-6 (IL-6 -174), and the tumor necrosis factor system (TNF-alpha -308 and lymphotoxin-alpha +252) were determined. Plasma C-reactive protein, leptin, and urinary pseudouridine (as a marker of cellular protein breakdown) were measured., Results: Fat mass, leptin, and pseudouridine were significantly different (P < 0.001) between noncachectic patients (NCPs) and cachectic patients (CPs: n = 35); the systemic inflammatory cytokine profile was not. NCPs had a body compositional shift toward a lower fat-free mass and a higher fat mass compared with HCs. CPs and NCPs had a greater systemic inflammatory response (P < 0.05) than did HCs, as reflected in C-reactive protein, soluble TNF-R75, and IL-6 concentrations. The overall distribution of the IL-1beta -511 polymorphism was significantly different between the groups (P < 0.05)., Conclusions: In COPD patients, who are characterized by an elevated systemic inflammatory response, cachexia is not discriminatory for the extent of increase in inflammatory status. This study, however, indicates a potential influence of genetic predisposition on the cachexia process.

Background: Weight loss indicates a poor prognosis in cystic fibrosis (CF). We hypothesised that fat-free mass (FFM) depletion and increased systemic inflammation would be associated with increased cellular proteolysis during an exacerbation of the respiratory symptoms. Patients were studied prospectively from the beginning of treatment with antibiotics when admitted to the Adults CF Centre., Methods: Twenty six patients with CF were studied at the start and end of antibiotic treatment and 2 weeks later. Mean (95% CI) FEV1 when clinically stable was 54.1 (44.5, 62.6)% predicted. Urinary excretion of Pseudouridine (5-ribosyluracil, PSU) was determined as an indicator of cellular protein breakdown. Body composition was assessed by dual energy X-ray absorptiometry (DXA)., Results: Patients had increased concentrations of PSU at all assessments (p<0.01). Those with a low FFM had greater PSU (ratio to FFMI) than those with a normal FFM at all assessments. At the start of treatment, PSU was related to FFM, C-reactive protein (CRP) (p<0.05) and tumour necrosis factor (TNF)alpha soluble receptors (sr) I and II (p<0.01). Circulating inflammatory mediators were greater in patients than in healthy subjects at all assessments., Conclusion: Increased protein breakdown is associated with a low FFM and increased systemic inflammation and it may be a contributory mechanism of poor weight preservation in CF.

A rapid, reliable method for the simultaneous determination of creatinine and pseudouridine is described. Both analytes were detected at an optimum wavelength of detection (262 nm), considering the relative levels present in bovine urine. Cimetidine was used as the internal standard and detected at its maximum wavelength of absorption (220 nm) on a separate channel. All three compounds were eluted within 15 min, using a 10 mmol/L phosphate buffer (pH 6.8)-methanol gradient on a C18 column. Creatinine data were found to be significantly dependent upon the pH of the sample. Recoveries of both analytes were above 96%. Lowest detectable levels of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, respectively. The use of internal standard resulted in a method with high precision (standard deviation of 1.42 mmol/L and 0.027 mmol/L for creatinine and pseudouridine), yet one that was simple and rapid.

Renally excreted modified RNA catabolites [pseudouridine (psi), N (2), N (2)-dimethylguanosine (m(2)(2)G) and N (6)-threoninocarbonyladenosine (t(6)A)] are markers of whole-body rates of degradation of rRNA and tRNA, and are thought to be potential indicators of the resting metabolic rate. To investigate diurnal variations of these RNA catabolites, the amounts of psi, m(2)(2)G and t(6)A excreted were determined by HPLC of the urine from eight healthy male adults collected over 47-h periods, which were subdivided into the morning (06.00 or 09.00 to 12.00 hours), the afternoon (12.00 to 18.00 hours), the evening (18.00 to 24.00 hours) and the night (00.00 to 06.00 or 08.00 hours), under two different nutritional regimens with 100 or 50 g of protein/day. Furthermore, rates of degradation of rRNA and tRNA were calculated using values for these RNA catabolites. For comparison, the corresponding excretion of creatinine, which originates from the energy metabolism of muscle, and of 3-methylhistidine (m(3)His), which is an indicator of muscle protein degradation, was determined. Differences in excretion during the collection periods were tested using the Friedmann test. The excretion of psi, creatinine and m(3)His (micromol x h(-1) x kg(-1)) altered significantly (P <0.001) during the day. Medians were: for psi, 0.21 (morning), 0.19 (afternoon), 0.19 (evening) and 0.18 (night); for creatinine, 8.8, 8.4, 8.0 and 7.3 respectively; for m(3)His, 0.13, 0.11, 0.12 and 0.10 respectively. The excretion rates of m(2)(2)G and t(6)A (nmol x h(-1) x kg(-1)) altered, but not significantly, during the day; corresponding medians were: for m(2)(2)G, 9.0, 8.4, 8.0 and 8.4 respectively; for t(6)A, 4.3, 4.1, 3.9 and 3.9 respectively. From these results it can be concluded that, in order to assess the average daily rates of degradation of tRNA and rRNA using modified RNA catabolites, urine collection should be carried out quantitatively over 24 h periods. Likewise, the catabolites creatinine and m(3)His must be determined using 24 h urine samples when average daily excretion values are required.

A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2'-deoxycytidine (m5dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 microg/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m5dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (psi), a modified nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary psi/Cre ratio was found significantly increased for leukaemia patients, the urinary m5dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease.

The elevation of urinary modified nucleosides levels in urine is found in patients with cancers. In the present study, we have tested 616 urine samples randomly collected from non-malignant cases. Thirty-two percent (194/616) and 11% (68/616) had elevated levels of 1-methyladenosine and pseudouridine, respectively (They are designated as false-positive cases). To elucidate the cause on non-specific elevation of the nucleosides, the correlation between creatinine excretion level and urinary nucleosides levels were determined. The result revealed that false-positive cases were frequently detected in patients with lower creatinine excretion levels. The mean creatinine levels of false-positive cases were significantly lower than those of negative cases. From these results, the false-positive of urinary 1-methyladenosine and pseudouridine might be due to the low creatinine excretion mainly caused by the renal dysfunction. Creatinine excretion in each individual should be taken into consideration in case of determining urinary modified nucleosides.

Determination of purine metabolites, pseudouridine and creatinine in both bovine and ovine urine using high-performance liquid chromatography (HPLC) is described. Following dilution and filtration, urine samples were analysed directly. Separation and quantification was achieved using a Spherisorb ODS II C18 column (250x4.6 mm I.D.) under isocratic conditions. The mobile phase contained 7.5 mM ammonium dihydrogen phosphate, 10 mM sodium 1-heptane sulphonic acid and 1.0 mM triethylamine at pH 3.0. Chromatography was achieved at a flow-rate of 1.0 ml/min and monitoring column effluent at 218 nm. Total analysis time was 60 min. Recovery of all compound standards added to urine was above 96%. In all cases, close spectral matches of compound standards and corresponding identified peaks in ovine and bovine urine were obtained. Lowest detectable concentrations of allantoin, uric acid, xanthine, hypoxanthine, creatinine and pseudouridine were 1.1, 1.0, 1.0, 3.0 and 0.4 micromol/l, respectively. Advantages of simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine collected from both sheep and cattle exist over current methods.

A simultaneous determination of uric acid, creatinine and pseudouridine in human urine by high performance capillary zone electrophoresis(HPCZE) is described. The urine samples were refrigerated and filtered. Then the samples were directly introduced into the capillary and a phosphate buffer solution(pH 6.1) was employed. The capillary used was 36 cm x 50 microns i.d. and detection was carried out with a UV monitor at 210 nm. The calibration curve showed a good linearity for uric acid, creatinine, and pseudouridine in the concentration range of 2-80 mg/L and their correlation coefficients were 0.996, 0.996 and 0.999 respectively. Within day CV for assaying the urine samples of uric acid, creatinine and pseudouridine were 5.0%, 3.0%, and 4.4%, and the corresponding between day data were 6.5%, 5.9%, and 5.3%, respectively. The recoveries of uric acid, creatinine and pseudouridine were 99.0%, 102.%, 92.2%, respectively. The total time for separation and determination was within 10 min. This method is characterized by direct assay of urine samples without any pretreatment. The results show that HPCZE is a simple, rapid, sensitive and reliable assay method.

In this paper a sensitive, rapid and simple HPLC method for the simultaneous determination of creatinine (Cr), pseudouridine (Pu) and uric acid(UA) has been established. We have evaluated clinical value of the method in the early diagnosis of diabetic nephropathy (DN). Separation was obtained using Shim-pack CLC-ODS 15 x 0.6 cm column and mobile phase of buffer solution of phosphate (pH 3.0, 0.02 mol/L) with flow rate of 1 mL/min. Detection was performed with UV detector at an automatic adjustment of wavelength. The recoveries of Cr, Pu and UA were 101.4%, 104.9% and 105.0% respectively. The calibration curves were linear within the concentration range of 8.6-274.6 mumol/L for Cr, 0.72-22.93 mumol/L for Pu and 19.1-612.7 mumol/L for UA (n = 6, r > 0.999, p < 0.01). The CV for within day and between day measurements were < 2.5% and < 5.0% respectively. In addition, Pu, Cr and UA were simultaneously determined in serum and urine of 39 patients with diabetic mellitus (DM). 15 patients with nephrotic syndrome (NS) and 53 normal subjects by the method. Results include: 1. the levels of serum Pu in all DM patients (100%) with microalbuminuria (MiAU) and macroalbuminuria (MaAU) were greater than maximum of normal subjects (X + 2S). In addition, in 9 DM patients (41%) with normal albuminuria (NAU) the levels were also greater than the maximum. 2. In patients with DM, there was no correlation between the serum Pu and the urinary albumin excretion (UAE), whereas the serum Pu correlated closely with the serum Cr. However, the incidence for serum Pu increase was significantly greater in patients. 3. The levels of serum Pu in patients with NS were greater than those in control subjects and patients with DM. Conclusion is that the determination of serum Pu could be used as sensitivity index for the early diagnosis of DN.

Background: There is a high degree of comorbidity between fibromyalgia and major depression. The latter is characterized by signs of immune activation, whereas the immune status in fibromyalgia is not yet elucidated. The aims of the present study were to examine (i) neopterin and biopterin excretion in 24-h urine of patients with fibromyalgia compared with normal volunteers and patients with major depression; and (ii) the effects of subchronic treatment with sertraline (11 weeks) on the urinary excretion of neopterin and biopterin., Methods: Measurements of neopterin, biopterin, pseudouridine, creatinine and uric acid in 24-h urine were performed by means of HPLC in 14 fibromyalgia and ten major depressed patients and 17 normal volunteers., Results: There were no significant differences in urine excretion of the above five analytes between patients with fibromyalgia and normal volunteers. Patients with major depression showed significantly higher urinary neopterin excretion than normal volunteers and fibromyalgia patients. Patients with fibromyalgia and major depression had a significantly increased neopterin/creatinine ratio. Fibromyalgia patients had significantly lower urinary excretion of creatinine than patients with major depression. In fibromyalgia patients, there were no significant effects of sertraline treatment on any of the urine analytes., Conclusions: The findings suggest that fibromyalgia, in contrast to major depression, may not be accompanied by activation of cell-mediated immunity., Limitation: Other immune markers should be measured in fibromyalgia before drawing definite conclusions., Clinical Relevance: Increased urinary excretion of neopterin can be used as a marker for major depression, but not fibromyalgia.

The levels of the five methylated nucleosides pseudouridine (psi-Urd), 1-methyladenosine (1-MeAdo), 4 acetylcytidine (4-AcCyd), 1 methylinosine (1-Melno) and 7 methylguanosine (7-MeGuo) resulting from RNA degradation were examined in the urine of rheumatoid arthritis (RA) patients. Of these five, 1-MeAdo and psi-Urd were correlated with the active phase of the disease, while two others (4-AcCyd and 1-Melno), which require further evaluation, appeared to be linked to the prognosis of the disease. As RNA turnover is closely associated with cell proliferation, including that of lymphocytes in RA, there may be a hitherto unsuspected benefit in measuring 1-MeAdo and psi-Urd as biochemical markers of RA disease activity.

We describe a capillary electrophoresis procedure, using uridine as an internal standard, for the analysis of urinary pseudouridine following solid-phase extraction. This method retains the advantages of existing chromatographic techniques but has superior resolving power and is technically less demanding. The standard curves were linear and reproducible with a detection limit of 60 fmol; chromatographic analysis was complete in under 10 min. Injection variability was < 5% and multiple independent analyses of the same urine sample for pseudouridine concentration gave coefficients of variation of < 10%. The mean (SD) urinary pseudouridine level in 18 healthy subjects was 16.1 (2.1) nmol/mumol creatinine. For a limited group of subjects where samples were taken more frequently, intra-individual variation averaged 27.5% reflecting variable excretion.

Pseudouridine, mainly as a degradation product of transfer ribonucleic acid, has been shown to be excreted in abnormal amounts in the urine of patients with cancer and can be used as one of the potentially valuable tumor markers. In this paper, a method for the rapid determination of human urinary pseudouridine by HPCE is reported. We used a 24 cm x 25 microm i.d. coated capillary and a borate buffer solution (0.1 mol/L, pH 8.6), and set the UV detection wavelength at 200 nm. In this experiment, pseudouridine can be separated completely with uridine and anti-tumor drugs (5-fluouridine and methetrexation) and other endogenous substances in urine within 4 minutes. Assay precision was determined for concentrations of 50 micromol/L and 200 micromol/L as 2.38% and 1.92% for within-day CV, 3.7% and 2.4% for between-day CV respectively. Sulfosalicylic acid was used as internal standard. Linearity, between the concentrations of pseudouridine and the corresponding peak height or area ratios of pseudouridine and the internal standard, was demonstrated in the 6.25-200 micromol/L range (r>0.9990). The detection limit of concentration for pseudouridine was 4 micromol/L. This method, with simple sample preparation, good assay precision, lower cost and full-automation, is an effective means for the assessment of pseudouridine in clinical application.

It is known that some modified, especially methylated, nucleosides originating from RNA degradation are excreted in abnormal levels in the urine of patients with malignant tumours and they have been proposed as tumour markers. Their measurement could provide a non-invasive diagnostic method, be helpful in the identification of different cancers and in the monitoring of therapeutic effects. In this study, we developed and optimized an analytical procedure to isolate and quantify normal and modified ribonucleosides. The extraction of urinary nucleosides was performed by affinity chromatography on a phenylboronic acid column prior to separation. The reversed-phase high-performance liquid chromatography method allowed a complete separation of sixteen urinary ribonucleosides. The recoveries for the different nucleosides ranged from 83 to 100%, except for xanthosine (66%) and pseudouridine (74%). In normal 24 h urine, the mean levels of thirteen nucleosides (in nmol of nucleoside/mumol of creatinine) were found to be as follows: dihydrouridine (6.37), pseudouridine (25.52), cytidine (0.07), uridine (0.21), 1-methyladenosine (2.19), inosine (0.30), guanosine (0.06), xanthosine (0.59), 3-methyluridine (0.11), 1-methylinosine (1.13). 1-methylguanosine (0.74), adenosine (0.21) and 5'-deoxy-5'-methylthioadenosine (0.12). The first results concerning two kinds of tumours, i.e. breast and floor of mouth tumours, showed some abnormal levels of ribonucleosides. Further experiments are now in progress to measure the modified nucleosides in urine of patients with different forms of cancer.

Unlabelled: The effect of daily human growth hormone (hGH) injections (3 I.U./m2/day) on tissue anabolism was determined in six patients with Ullrich-Turner syndrome (XO) (8.7-19 years of age) using novel techniques such as whole body leucine kinetics during continuous infusion of L-(Methyl-2H3)-leucine and urinary pseudouridine (5-ribosyluracil) excretion on the one hand and traditional methods like serum urea and amino acid concentrations on the other. Pseudouridine is only found in ribonucleic acid (RNA) and is neither reincorporated nor catabolically broken down and is therefore considered an ideal index of whole body RNA turnover. The mean L-(Methyl-2H3)-leucine turnover of the six XO patients before hGH was 1.90 +/- 0.15 mumoles/kg per minute. After 3 months of hGH-treatment it had increased in three patients, whereas it had decreased in the other three. The results obtained with the stable isotope technique were correlated with the urinary pseudouridine concentrations (r = 0.68; P < 0.01). The growth rates were positively correlated with leucine turnover (r = 0.63; P < 0.02) and urinary pseudouridine concentration (r = 0.73; P < 0.006) as well as negatively correlated with the serum urea concentrations r = -0.62; P < 0.03). The decrease in the individual serum urea concentrations were tightly correlated with the hGH induced change in growth rate (r = -0.90; P < 0.01). The individual bone ages were negatively correlated with the hGH induced changes in leucine turnover (r = -0.77; P < 0.003) as well as with the urinary pseudouridine concentrations (r = -0.87; P < 0.0002). The hGH effect on leucine and RNA turnover, showing effectiveness only until a developmental age between 11 and 12 years, leads the discussion of the ideal moment of oestrogen supplementation when girls with Ullrich-Turner syndrome are treated with hGH in early adolescence., Conclusion: The protein metabolism of patients with Ullrich-Turner syndrome is influenced by hGH in an age dependent manner. In a clinical setting, pseudouridine, an easily determined derivative of ribonucleic acids, may be able to replace the tedious work with expensive stable isotopes when questions related to tissue anabolism are to be answered.

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.

A simple, fast and precise method for the simultaneous determination of neopterine (Npt), pseudouridine (Psu) and creatinine (Cre) in urine by using ion-pair HPLC has been developed. The urine specimen is subjected to a microcolumn clean-up step, providing a ten-fold sample dilution, and is then injected into the column. The eluate is forced through the respective cells of a fluorescence and then a UV detector, which are connected in series. Neopterine is monitored by fluorescence emission at 438 nm (excitation wavelength 353 nm) while Psu and Cre are monitored by UV absorption at 235 nm. This allows the determination of Npt/Cre and Psu/Cre concentration ratios in a single run on the same sample, and the use of urine specimens collected randomly instead of 24 hourly collections. The method has been applied to urine specimens from 19 healthy, male donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mmol) values of 41.7 and 16.8, respectively) and 23 healthy, female donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mol) values of 67.4 and 18.5, respectively). The within-run S(r) for Npt/Cre and Psu/Cre concentration ratios ranged between 3.3 and 4.6%.

It was demonstrated earlier that urine of rats bearing transplanted mesoblastic nephroma had high mutagenic activity in Salmonella typhimurium, which could not be detected in serum samples of the same animals. In this paper, cytogenetic alterations are discussed and the lack of enhanced micronucleus formation in bone marrow of tumorous rats is described. The cytogenetic effect of the hydrophobic (XAD-4) urinary fraction, which has been found to be mutagenic in the TA98 Salmonella strain, was examined in CBA mice. Sister chromatid exchange (SCE) analyses were performed on bone marrow cells of animals treated with single injections of concentrated urine samples. Significant and continuous increases could be detected in the SCE frequencies caused by the urinary concentrates with development of the tumour. Pseudouridine, a suggested urinary tumour marker nucleoside, was also studied for mutagenicity in the Ames Salmonella test. Both derivatives (alpha and beta), however, failed to induce mutations in the TA98/TA100 strains, either with or without metabolic activation. In conclusion, urinary mutagen(s) produced during the renal tumour growth have a spectrum of genotoxicity involving at least two endpoints, but the high pseudouridine excretion may not be responsible for these effects.

The present experiment was carried out to study the endogenous losses of purine and pyrimidine derivatives from pregnant sows. Three pregnant and three non-pregnant Large White x Landrace sows were fed on a purine-free diet composed of starch, glucose, sucrose and vegetable oil, with casein as the protein source. The experiment began, for the six animals, after diagnosis of pregnancy and was divided into six 12 d periods. Urine was collected during the first 3 d of each experimental period by means of a urethral catheter for determination of allantoin, uric acid, xanthine, hypoxanthine and pseudouridine concentrations. In the absence of dietary nucleic acids (NA), allantoin and, as a consequence, excretion of total purine derivatives (PD) decreased significantly to a constant value (128.3 (SE 7.07) mumol/kg metabolic live weight (W0.75) per d), an amount assumed to represent endogenous excretion. Excretion of uric acid (38.7 (SE 2.15) mumol/kg W0.75 per d), hypoxanthine (21.0 (SE 2.58) mumol/kg W0.75 per d) and xanthine (11.2 (SE 0.83) mumol/kg W0.75 per d) were not affected by the experimental treatment, although there was a significant decrease in hypoxanthine excretion in pregnant sows (from 25.5 to 5.2 mumol/kg W0.75 per d) compared with non-pregnant sows (from 26.7 to 44.8 mumol/kg W0.75 per d). Creatinine excretion was not affected by pregnancy and was used as an internal urinary marker. Purine excretion, either expressed as mumol/kg W0.75 per d or as the ratio PD: creatinine, was not affected by experimental treatment, although an apparent increase in pseudouridine excretion, a modified unsalvageable catabolite of RNA-pyrimidine, was found in late pregnancy (3.6 v. 5.2 mol/100 mol creatinine in non-pregnant sows compared with pregnant sows at 102 d collection.

Urinary and serum pseudouridine concentrations were determined by high-performance liquid chromatography in 80 patients with primary liver cancer, 32 with benign space occupying lesions of the liver, 42 with liver cirrhosis and 40 healthy subjects. Their mean urinary and serum pseudouridine levels were 39.2 +/- 11.5 nmol/mumol creatinine and 3.4 +/- 1.3 mumol/L, 24.5 +/- 5.4 nmol/mumol creatinine and 2.5 +/- 0.5 mumol/L, 22.8 +/- 7.8 nmol/mumol creatinine and 2.3 +/- 0.4 mumol/L, 26.4 +/- 4.6 nmol/mumol creatinine and 2.3 +/- 0.4 mumol/L, respectively. Exceeding the mean plus 2SD of pseudouridine of healthy control was considered as positive value for the diagnosis of primary liver cancer. Thus the positivity of urinary and serum pseudouridine in hepatoma was 71.3% and 70.0%, respectively. The positive rate of combined pseudouridine and alpha-fetoprotein assay was 91.3% in patients with hepatoma. Besides, pseudouridine levels could elevate before positive localization and reduce to normal levels after tumor resection. The results showed that the determination of pseudouridine is of clinical significance in the diagnosis and monitoring of primary liver cancer.

Urinary excretion of pseudouridine, a modified nucleoside, was assessed in 30 patients with Hodgkin's disease, and 106 patients with non-Hodgkin's lymphoma, classified according to the Kiel system. Elevated excretion was found in 47% of 49 patients with high-grade malignant (HGM) lymphoma, and in 37% of 57 with low-grade malignant (LGM) lymphoma, in 13% in Hodgkin's disease, and 3% in 79 reference individuals. The level of pseudouridine excretion correlated with clinical stage in HGM lymphoma (p < 0.0001), but not in LGM lymphoma or Hodgkin's disease (p = 0.086 and 0.36 respectively). Of 28 patients with B-symptoms 71% had elevated excretion, compared to 26% of 108 without B-symptoms (p < 0.0001). Elevated excretion of pseudouridine before therapy was associated with shorter survival time in LGM lymphoma stage II to IV disease, (p = 0.022), and a similar tendency was also observed in HGM lymphoma. Using Cox proportional hazard model, age, malignancy grade, excretion of pseudouridine, and disease stage were identified as independent prognostic factors in non-Hodgkin's lymphoma.

The urinary concentration of pseudouridine, primarily a breakdown product of tRNA, was determined by high-performance liquid chromatography in 33 patients with small cell lung cancer (SCLC), 18 patients with non-small cell lung cancer (NSCLC), 18 patients with benign pulmonary diseases (eg, TB, COPD), and 16 healthy controls. The mean urinary pseudouridine concentration levels of the patients with SCLC, NSCLC, benign pulmonary disease and the healthy controls were 22.7 +/- 11.8, 15.6 +/- 6.0, 13.4 +/- 3.6, 12.1 +/- 3.2 nmol/mumol creatinine (mean +/- SD), respectively. The mean urinary pseudouridine concentration was significantly higher in patients with SCLC than that in patients with NSCLC, benign pulmonary disease and in the healthy controls. There was no significant difference in the urinary pseudouridine levels of patients with NSCLC and healthy controls or patients with benign pulmonary disease. Urinary pseudouridine levels above 18.5 nmol/mumol creatinine (mean value +/- 2SD for healthy controls) were noted in 54.5% of patients with SCLC, including 11 of 17 (64.7%) in extensive stage and seven of 16 (43.8%) in limited stage, and 27.8% of the patients with NSCLC. Of the 12 patients with SCLC who had follow-up urinary samples taken in series during chemotherapy courses, there was considerable urinary pseudouridine level change which paralleled the change in clinical response. Although urinary pseudouridine is not a specific marker for SCLC, it represents the tumor burden and reflects the clinical status. These findings indicate that urinary pseudouridine may be a useful tumor marker in patients with SCLC.

Background: Modified nucleosides such as 1-methyl-adenosine and pseudouridine exist as minute components of transfer ribonucleic acid (tRNA) and are excreted in the urine in large amounts in the presence of malignancy. Although use of these modified nucleosides as tumor markers has long been studied and many reports have detailed their relationship with malignant tumors and the urinary excretion of various modified nucleosides, there have been no reports on modified nucleosides in esophageal carcinoma., Methods: Monoclonal antibody patterns against 1-methyladenosine and pseudouridine were studied in esophageal carcinoma, freshly resected esophageal carcinoma tissue specimens fixed in 10% neutral formaldehyde solution, embedded in paraffin, and sectioned for immunohistochemical study. Inhibition enzyme-linked immunosorbent assay (ELISA) was used to examine urinary excretion of these modified nucleosides in patients with esophageal carcinoma., Results: Although rare in normal esophageal epithelium, these modified nucleosides were strongly stained in esophageal carcinoma cells. Most carcinoma cells exhibited a cytoplasmic pattern, although some cells at the infiltrating edge displayed a nuclear pattern. These modified nucleosides were intensely imaged in 11 of 12 cultured esophageal cell lines, the exception being one line that had a much longer doubling time. Using ELISA, urinary excretion of these modified nucleosides was found to be significantly higher in patients with esophageal carcinoma than in healthy subjects; such excretion correlated with carcinoma size and stage and tended to decrease after treatment., Conclusions: These findings indicate that the modified nucleosides 1-methyladenosine and pseudouridine may be useful as tumor markers for esophageal carcinoma.

Random urine samples from 614 neonates were screened for metabolites of purine and pyrimidine metabolism using an adapted column chromatographic method. A restricted number of metabolites and a number of unidentified peaks appeared on the chromatograms. No inborn errors of this metabolism were found. The chromatograms were identical in term and in premature or dysmature neonates, except for the presence of more unidentified peaks in the latter group. The pattern was not influenced by the type of feeding or i.v. nutrition. Metabolites of different medications were identified. One female neonate with an increased excretion of uracil was shown to be heterozygous for ornithine carbamyl transferase deficiency.

The blood plasma concentration of pseudouridine was estimated in 104 healthy adult subjects, and 108 patients suffering from malignant proliferative diseases. The HPLC method for simultaneous determination of pseudouridine and creatinine was applied. The average physiological concentration of pseudouridine in blood plasma was 2.43 +/- 0.97 mumol.l-1 or 29.15 +/- 7.40 mmol.mol-1 creatinine. The physiological urinary excretion of pseudouridine was 14.32 +/- 5.20 mumol.24 h-1.kg-0.75 or 19.60 +/- 5.22 mmol.mol-1 creatinine. Renal clearance of pseudouridine and endogenous creatinine were 4.04 +/- 0.99 and 5.50 +/- 1.46 ml.kg-0.75, respectively. A positive correlation (r = 0.55, P < 0.01) was found between age (in the range 20-92 years) and blood plasma pseudouridine concentration (mumol.l-1). By expressing plasma pseudouridine in relation to plasma creatinine, the apparent influence of non-metabolic factors (age, renal insufficiency, blood dilution) on the plasma pseudouridine concentration were largely excluded. Among haematological proliferative diseases the highest values of plasma pseudouridine concentrations were observed in chronic lymphocytic leukaemia (8.19 mumol.l-1; 54.9 mmol.mol-1 creatinine) and multiple myeloma (7.02 mumol.l-1; 52.5 mmol.mol-1 creatinine). In multiple myeloma, but not in chronic lymphocytic leukaemia, the plasma pseudouridine concentration depended on the clinical stage. A lower, but still significant response in non-Hodgkin's lymphoma was noted (4.03 mumol.l-1; 40.88 mmol.mol-1 creatinine). A significant increase of the plasma pseudouridine concentration was characteristic of adenocarcinomas of the large intestine, and it occurred in the early stages of malignant growth. In patients with lung cancer the plasma pseudouridine concentration was elevated only in advanced cases with metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

The HPLC method for the simultaneous determination of urinary neopterin, pseudouridine, and creatinine allows a rapid evaluation of the activation state of cell-mediated immunity, and the stimulation of whole-body rRNA + tRNA turnover, associated with malignant growth. Urinary neopterin and pseudouridine concentrations in healthy subjects amounted to: 106.6 +/- 34.6 mumol/mol creatinine, and 19.6 +/- 5.2 mmol/mol creatinine (mean +/- SD), respectively. The increase of neopterin excretion in patients with haematological neoplasms ranged from 146% in Hodgkin's disease to 534% in non-Hodgkin's lymphoma, whereas the increase in cancer cases ranged from 95% in adenocarcinoma of the gaster to 741% in hepatocellular carcinoma. The changes in pseudouridine excretion were much less pronounced: 63% in non-Hodgkin's lymphoma and 120% in carcinoma of the urinary bladder. The correlation coefficient between neopterin and pseudouridine was relatively low (r = 0.43), although statistically significant (P < 0.01). In the case of several neoplasms e.g. Hodgkin's disease, polycythaemia vera, and adenocarcinoma of the gaster, neopterin was significantly elevated, whereas pseudouridine remained at a normal concentration. There was a positive relationship between the stage of the disease (primary focus, regional metastases, dissemination) and urinary concentration of pseudouridine in patients with adenocarcinoma of the large intestine. In the same patients the increase of neopterin excretion was noticed both in early and advanced stages, with the highest values in disseminated disease.

Elevated urinary levels of modified nucleosides, especially pseudouridine (psi), have been observed in patients with a variety of malignant neoplasms. In this report, the urinary psi levels were estimated by high performance liquid chromatography in 93 patients with brain tumours and in 40 age and sex matched controls. The psi levels were found to be excreted in equal amounts in the control and the tumour groups. There was no significant difference in psi when patients with malignant diseases were compared with those with benign tumours. Following therapy, the psi levels remained unchanged when compared to the controls and their preoperative values. It is concluded that urinary psi concentration is not a useful marker for brain tumours.

The preoperative and postoperative values of urinary pseudouridine:creatinine (phi:C) and beta-aminoisobutyric acid:creatinine (beta AIB:C) were estimated, in 192 patients with urothelial tumours of the bladder, 92 of whom had not previously been diagnosed. Urinary phi:C ratio correlated with the grade of tumour cell dysplasia (being highest in dysplasia grade 3), and to a lesser extent with the clinical stage. The treatment had no major influence on the excretion ratios. Decreased ratios, or those within the reference range, were associated with a better prognosis than increased ratios, and if both were increased at the same time the risk for progression of the disease was high. The biological tumour markers pseudouridine and beta-aminoisobutyric acid may be helpful in the diagnosis of tumours in the upper urinary tract, and in the follow-up of patients with tumours of the bladder.

By use of monoclonal antibodies (MoAbs) termed APU-6 and AMA-2, we determined the usefulness of urinary modified nucleosides, pseudouridine and 1-methyladenosine, as markers for malignancy. In patients with leukemia and other forms of cancer, these nucleosides elevated significantly and reflected the disease status of patients. The immunohistochemical analysis showed that cancer cells were specifically stained with the MoAbs. Chemical identification of the cellular components reactive with the MoAbs revealed that APU-6-associated antigens were mainly rRNA and AMA-2-associated antigens were mainly tRNA. These results suggest that APU-6 and AMA-2 would be useful tools for clinical and biological studies of cancer.

A reversed phase high performance liquid chromatographic method for the simultaneous determination of pseudouridine (PU) and creatinine (Cr) in urine is described. The mobile phase was 0.01 mol phosphate buffer (pH 6.1) containing 2.5 mmol octanesulphonic acid as the ion pairing agent. UV detection was set at 250 nm. Variation in pH value affected the retention time of PU and Cr significantly; Their separation from interfering peaks was also affected. The recoveries of PU and Cr were 89.93% and 90.35%, respectively. The standard deviation of the method for PU was 48.69 +/- 0.063 (nmol/mumol Cr, mean +/- SD, n = 5). The urine samples from 233 normal children of different ages and 119 patients with leukaemia were analysed by this method. The normal reference value was appraised by comparison with the percentage of immature cells in the bone marrow. The results showed that the sensitivity of the method was 94.12%, the specificity was 95.86%, the accuracy was 95.50%, the positive predictive value was 82.05% and the negative predictive value was 98.78%. The method can be used to evaluate the state of the leukaemia, and to monitor the effect of treatment.

Urinary levels of pseudouridine and 1-methyladenosine in patients with leukemia and lymphoma were measured by the inhibition ELISA using monoclonal antibodies to determine the correlation of nucleosides excretion with disease activity. Significantly elevated levels of these nucleosides were detected in patients with all types of disease tested. Seventy-seven percent (46/60) and 62% (37/62) of patients had elevated levels of pseudouridine and 1-methyladenosine above normal mean + 2S.D., respectively, and combination assay of these nucleosides gave higher positive rate (87%; 52/60) than either single assay. The changes of urinary pseudouridine and 1-methyladenosine reflected the disease status of patients in remission or in relapse and the effect of chemotherapy. These results suggest that urinary pseudouridine and 1-methyladenosine might be clinically useful as complementary markers to the monitoring of the disease status of patients with leukemia and lymphoma by hematological examination.

Pseudouridine (psi) is a unique nucleoside accumulated in the sera of renal failure (RF) patients. Surprisingly data on its excretion are lacking. To get an overview, the psi serum level and urinary excretion were investigated in 73 healthy subjects (C), 16 patients not on dialysis (ND) and 12 hemodialysis patients (D). It was found: (a) psi accumulates in the sera of both ND and D patients. An inverse power correlation fits best with the relationship between serum psi and the clearance of endogenous creatinine (CCr). The amount of psi filtered in glomeruli of ND patients increases while it remains practically unchanged in D patients. However, the psi filtration load of residual nephrons increases with the decreasing CCr as a consequence of its increased serum concentration. (b) Both psi net resorption and secretion have been found in C subjects. The increased psi resorption diminishes the necessary increase of psi urinary excretion both in ND and D patients. The increase of psi resorption is marked if calculated on residual nephrons. (c) The slightly decreased psi excretion excludes the participation of its increased synthesis in its accumulation in RF. It is concluded that psi accumulation in RF is caused by the impairment of its kidney excretion and the increased psi resorption participates markedly in its retention.

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5-20 microliters samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 +/- 14.4 mumol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.

Pseudouridine is the most prevalent modified nucleoside excreted in urine, mainly as a degradation product of t-RNA. The level of pseudouridine excretion was analyzed in 71 patients with renal cell carcinoma prior to treatment. An increased excretion was demonstrated in 27 of 48 patients (56%) in stage II-IV, compared to 2 of 23 patients (9%) in stage I. Survival time was significantly reduced in patients with increased excretion. The level of pseudouridine correlated to tumor grade and tumor size. Using Cox's proportional hazard model, only clinical stage and level of pseudouridine excretion were independent predictors of prognosis.

The catabolism of tRNA gives rise to modified nucleosides, among which pseudouridine, which are excreted in urine. An increased cell turnover is associated with an enhanced excretion of these products. Some authors have also reported an increased urinary excretion of pseudouridine in asbestos workers without clinical signs of malignancy. In two experimental models of carcinogenesis (i.e. injection of Lewis carcinoma cells in C57/B1C mice and oral administration of 7,12 dimethyl-benz(a)anthracene in female Sprague-Dawley rats), we have confirmed that the occurrence of the tumor is associated with an increased urinary excretion of pseudouridine. This metabolic change, however, does not precede the detection of the tumor. In control human subject, no sex and age effect was found in the urinary concentration of pseudouridine. Patients with a malignant disease excrete an enhanced amount of pseudouridine. A pilot study among workers exposed to polycyclic aromatic hydrocarbons in a coke oven did not reveal any change in the urinary concentration of pseudouridine.

Modified nucleosides from pooled normal human urine were extracted using a boronate affinity gel column and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The major constituents in each of the 30 RP-HPLC fractions were determined by gas chromatography-mass spectrometry of the trimethylsilyl derivatives of the fractions. The same RP-HPLC method was used in the synthesis of 5'-deoxyxanthosine from authentic 5'-deoxyadenosine. In addition, the simultaneous determination of urinary 5,6-dihydrouridine (D) and pseudouridine (psi) was carried out by RP-HPLC using two ODS columns in series. The level of D in pooled normal urine was 4.87 nmol/mumols creatinine. The RP-HPLC method was applied to the measurement of D and psi levels in urines collected before and after surgery from four patients with gastrointestinal cancer. A large decline in both nucleoside levels in urines after surgery was observed in three of the four cancer patients.

In 39 patients with either pelvic or renal cell tumours, the pre-operative urinary excretion ratios of pseudouridine/creatinine (psi:C) and beta-aminoisobutyric acid/creatinine (BAIB:C) were estimated. In 34/39 of the patients (87%), including both types of tumours, psi: C was found to be increased, and in only one patient was the value decreased. Twenty-five of the patients (64%) had a BAIB:C ratio within the normal range. 10 (25%) had increased and 4 (11%) decreased values. In 13 patients the psi:C and BAIB:C ratios from the affected side were compared with the values in urine from the bladder. Twelve of the patients had higher values of psi:C in the urine from the ureter. In seven patients the ureteric BAIB:C ratio was higher than the bladder BAIB:C ratio and in five patients the ureteric BAIB:C ratio was less than the bladder BAIB:C ratio. In only one patient were the values similar. The results indicate that patients with tumours of the upper urinary tract have changed excretion patterns compared with normal subjects and the urinary ratios on the affected side are different from those on the healthy side. The biological tumour markers pseudouridine and beta-aminoisobutyric acid may be helpful in the diagnosis of tumours in the upper urinary tract.