Urinary chemical fingerprint left behind by repeated NSAID administration: Discovery of putative biomarkers using artificial intelligence.

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.

From Discovery to Translation: Characterization of C-Mannosyltryptophan and Pseudouridine as Markers of Kidney Function.

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 (25th, 75th 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.

Tailor-Made Molecularly Imprinted Polymer for Selective Recognition of the Urinary Tumor Marker Pseudouridine.

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.

A Novel Urinary Metabolite Signature for Non-invasive Post-stroke Depression Diagnosis.

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.

Development and validation of a method for simultaneous determination of pseudouridine and creatinine in human urine. Evaluation’s Index Pseudouridona/Creatinine in smokers and no-smokers

Introduction: The excretion of pseudouridine is increased in inflammatory processes related to a muscle mass loss found in patients with pulmonary involvement. Material and Methods: A rapid and sensitive method for cuantification and simultaneous determination of pseudouridine, a breakdown product of tRNA, and creatinine in human urine via HPLC was developed and validated. The mobile phase was 0.01 mol phosphate buffer (pH 6.1) containing 3 mmol octanesulphonic acid as ion pairing agent. Sample preparation is based on dilution and filtration. A LiCHros-pher® 100 RP-18 (5 μm) LiCHroCART® 250-4 (Merck) column with precolumn LiCHrospher® 100 RP-18 (5 μm) LiCHroCART® 50-4 (Merck) were used, flow rate of 1ml/min. Detection wavelength was set at 250 nm. Results: The analysis time was 17 min per sample. The calibration range of pseudouridine (Psu) and creatinine (Crea) were 0.23-22.5 and 11.45-1100 nmol/ml. The linearity of the method was r2 = 0.997 and 0.998 and the lower limit of quantification (LLOQ) was 0.175 and 8.59 nmol/ml respectively. The average recovery (%) was 95.55 for pseudouridine and 97.82 for creatinine by addition and 93.16 and 89.79 % by dilution. The estimation of the coefficients of variation were < 8% for all levels. Conclusions: A positive correlation was found between expected and observed values (Pearson correlation coefficient = 0.99 for pseudouridine and 0.99 for creatinine). A correlation was found between recovery of pseudouridine and recovery of creatinine (Pearson correlation coefficient = 0.86). This method was used to assess pseudouridine excretion in 30 healthy subjects (18 non-smokers and 12 smokers). There were no statistically differences between non-smokers and smokers (AU)

Introdución: La excreción de pseudouridina, está incrementada en procesos inflamatorios relacionados con pérdida de masa muscular encontradaen pacientes con afectación pulmonar. Material y Métodos: Se desarrolla y valida, un método, mediante HPLC, para la determinación simultanea de pseudouridina y creatinina en orina. Como fase estacionaria se utilizó una columna LiCHrospher® 100 RP-18 (5 μm) LiCHroCART® 250-4 (Merck). Fase móvil consistente en un tampón fosfato 0,01 M (pH = 6,1) conteniendo octanosulfónico 3mmol como agente de par iónico, y longitud de onda de 250 nm. La preparación de la muestra se basa en una dilución y filtración. La linealidad del método fue satisfactoria dentro del rango de concentración de 0,23-22,5 nmol/ml para pseudouridina y 1,45-1100 nmol/ml para creatinina, con límites de cuantificación de 0,175 y 8,59 nmol/ml, respectivamente. Resultados: La recuperación media fue del 95,55% para Pseudouridina y del 97,82% para Creatinina en la validación de adición de estándar interno y de 93,16 y 89,79% en la de dilución. Los coeficientes de variación fueron < del 8% en todos los niveles. Se encontró una correlación entre los valores encontrados y los esperados (coeficiente de correlación de Pearson de 0,99). Conclusiones: Existe correlación entre las recuperaciones de pesudouridina y creatinina, coeficiente de correlación de Pearson de 0,86. El método desarrollado, es rápido, sensible y selectivo para la simultanea determinación de pseudouridina y creatinina en orina humana. En este estudio preliminar con 30 voluntarios sanos, 18 no fumadores y 12 fumadores, no se han encontrado diferencias estadísticamente significativas entre ambos grupos con respecto a la excreción de pseudouridina (AU)

[Muscle catabolism in patients with chronic obstructive pulmonary disease]. / Catabolismo muscular en pacientes con enfermedad pulmonar obstructiva crónica.

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.

Análisis del catabolismo muscular en la enfermedad pulmonar obstructiva crónica / Analysis of the muscle catabolism in chronic obstructive pulmonary disease

Introducci¨®n: La enfermedad pulmonar obstructiva cr¨®nica (EPOC) es la cuarta causa de muerte en el mundo, en la que se aprecia afectaci¨®n muscular, en estadios iniciales asintom¨¢ticos, producto del desequilibrio proteico que ha sido medido en otros procesos mediante la excreci¨®n urinaria de pseudouridina. Objetivos: Investigar, mediante la excreci¨®n urinaria de pseudouridina, si el catabolismo muscular en EPOC est¨¢ elevado, y en qu¨¦ medida se relaciona con la edad y con el sexo. Material y m¨¦todos: El estudio se basa en un an¨¢lisis prospectivo y aleatorio de una poblaci¨®n control y otra con EPOC. La determinaci¨®n simult¨¢nea de pseudouridina y creatinina en orina se lleva a cabo por cromatograf¨ªa l¨ªquida de alta resoluci¨®n (HPLC). Se realiza un estudio estad¨ªstico descriptivo de las variables edad, sexo, excreci¨®n de pseudouridina y, mediante curva ROC de la variable pseudouridina excretada, se determina el punto de corte con mayor sensibilidad y especificidad entre ambas poblaciones. Resultados: No se observan diferencias estad¨ªsticamente significativas en la pseudouridina excretada, ni con respecto a la edad ni al sexo, en los grupos estudiados. La excreci¨®n de pseudouridina fue significativamente mayor en los pacientes con EPOC, 94,40 (87,64) ¦Ìmol/mmol con respecto al grupo control, 19,90 (6,6) ¦Ìmol/mmol Md (IQR) (p < 0,001). En el an¨¢lisis mediante curva ROC, el punto de corte ¨®ptimo con mayor sensibilidad y especificidad para distinguir personas con un catabolismo muscular alterado es el correspondiente a una excreci¨®n de pseudoridina de 29,39 ¦Ìmol/mmol. Conclusiones: La medida de la pseudouridina excretada en orina, realizada con este m¨¦todo,puede ser utilizada como control del catabolismo muscular. En nuestro estudio, el ¨¢rea bajo la curva para la excreci¨®n urinaria de pseudouridina fue de 0,972 y el punto de corte obtenido, fue 29,39 ¦Ìmol/mmol y corresponder¨¢ a individuos con un catabolismo incrementado debido a la EPOC (AU)

Introduction: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the world, appreciating muscle involvement, asymptomatic in early stages, which is due to a protein imbalance that has been measured in other processes by the urinary excretion of pseudouridine. Objectives: To research, using urinary excretion of pseudouridine, if muscle catabolism in COPD is high and until what extent is related to age and gender. Material and methods: The study is based on an analysis of a prospective randomized control group and one with COPD. Simultaneous determination of pseudouridine and creatinine in urine was performed by high-performance liquid chromatography (HPLC). We performed a descriptive statistical study of the variables age, gender, excretion of pseudouridine, and a ROC curve of the variable pseudouridine excretion, determines the cut point with higher sensitivity and specificity between the two populations. Results: No statistically significant differences were found in the pseudouridine excreted, with respect to age or gender, in the groups studied. Pseudouridine excretion was significantly higher in patients with COPD, 94.40 (87.64) ¦Ìmol/mmol, with respect to the control group, 19.90 (6.6) ¦Ìmol/mmol Md (IQR) (p <0.001). In the ROC curves analysis, pseudouridine excretion had area under the curve 0.972 and the optime cut point for pseudouridine excretion in our trial was 29.39 ¦Ìmol/mmol (Sensibility 93.1% and Specificity 100%) and can be used to distinguish people with altered muscle catabolism. Conclusions: The measure of pseudouridine excreted in urine, performed by this method, can be used as a control of muscle breakdown. In our study, the cut point was 29.39 ¦Ìmol/ mmol which means, that people with a pseudouridine excretion 29.39 ¦Ìmol/mmol, shall be individuals with an increased catabolism due to COPD (AU)

Noninvasive markers of oxidative DNA stress, RNA degradation and protein degradation are differentially correlated with resting metabolic rate and energy intake in children and adolescents.

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.

Cellular protein breakdown and systemic inflammation are unaffected by pulmonary rehabilitation in COPD.

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.

Urinary pseudouridine in patients with lymphoma: comparison with other clinical parameters.

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.

Pulmonary cachexia, systemic inflammatory profile, and the interleukin 1beta -511 single nucleotide polymorphism.

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.

Cellular proteolysis and systemic inflammation during exacerbation in cystic fibrosis.

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.

Simultaneous liquid chromatographic determination of creatinine and pseudouridine in bovine urine and the effect of sample pH on the analysis.

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.

Diurnal variation in the renal excretion of modified RNA catabolites in humans.

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.

Creatinine at the evaluation of urinary 1-methyladenosine and pseudouridine excretion.

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.

Simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine by reversed-phase high-performance liquid chromatography.

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.

Liquid chromatographic determination of urinary 5-methyl-2'-deoxycytidine and pseudouridine as potential biological markers for leukaemia.

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.

Immune markers in fibromyalgia: comparison with major depressed patients and normal volunteers.

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.

[Simultaneous determination of creatinine, pseudouridine and uric acid in serum and urine by high performance liquid chromatography].

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.