Metabolite Biomarkers of CKD Progression in Children.

BACKGROUND AND OBJECTIVES: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR. RESULTS: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m2; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m2, two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m2, a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9). CONCLUSIONS: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways.

Pseudouridine and N-formylmethionine associate with left ventricular mass index: Metabolome-wide association analysis of cardiac remodeling.

BACKGROUND: Heart failure (HF) is the fastest growing form of cardiovascular disease both nationally and globally, underlining a need to phenotype subclinical HF intermediaries to improve primary prevention. OBJECTIVES: We aimed to identify novel metabolite associations with left ventricular (LV) remodeling, one upstream HF intermediary, among a community-based cohort of individuals. METHODS: We examined 1052 Bogalusa Heart Study participants (34.98% African American, 57.41% female, aged 33.6-57.5 years). Measures of LV mass and relative wall thickness (RWT) were obtained using two-dimensional-guided echocardiographic measurements via validated eqs. LV mass was indexed to height2.7 to calculate left ventricular mass index (LVMI). Untargeted metabolomic analysis of fasting serum samples was conducted. In combined and ethnicity-stratified analyses, multivariable linear and multinomial logistic regression models tested the associations of metabolites with the continuous LVMI and RWT and categorical LV geometry phenotypes, respectively, after adjusting for demographic and traditional cardiovascular disease risk factors. RESULTS: Pseudouridine (B = 1.38; p = 3.20 × 10-5) and N-formylmethionine (B = 1.65; 3.30 × 10-6) were significantly associated with LVMI in the overall sample as well significant in Caucasians, with consistent effect direction and nominal significance (p < .05) in African Americans. Upon exclusion of individuals with self-report myocardial infarction or congestive HF, we similarly observed a 1.33 g/m2.7 and 1.52 g/m2.7 higher LVMI for each standard deviation increase in pseudouridine and N-formylmethionine, respectively. No significant associations were observed for metabolites with RWT or categorical LV remodeling outcomes. CONCLUSIONS: The current analysis identified novel associations of pseudouridine and N-formylmethionine with LVMI, suggesting that mitochondrial-derived metabolites may serve as early biomarkers for LV remodeling and subclinical HF.

A Prospective Analysis of Circulating Plasma Metabolites Associated with Ovarian Cancer Risk.

Ovarian cancer has few known risk factors, hampering identification of high-risk women. We assessed the association of prediagnostic plasma metabolites (N = 420) with risk of epithelial ovarian cancer, including both borderline and invasive tumors. A total of 252 cases and 252 matched controls from the Nurses' Health Studies were included. Multivariable logistic regression was used to estimate ORs and 95% confidence intervals (CI), comparing the 90th-10th percentile in metabolite levels, using the permutation-based Westfall and Young approach to account for testing multiple correlated hypotheses. Weighted gene coexpression network analysis (WGCNA; n = 10 metabolite modules) and metabolite set enrichment analysis (n = 23 metabolite classes) were also evaluated. An increase in pseudouridine levels from the 10th to the 90th percentile was associated with a 2.5-fold increased risk of overall ovarian cancer (OR = 2.56; 95% CI, 1.48-4.45; P = 0.001/adjusted P = 0.15); a similar risk estimate was observed for serous/poorly differentiated tumors (n = 176 cases; comparable OR = 2.38; 95% CI, 1.33-4.32; P = 0.004/adjusted P = 0.55). For nonserous tumors (n = 34 cases), pseudouridine and C36:2 phosphatidylcholine plasmalogen had the strongest statistical associations (OR = 9.84; 95% CI, 2.89-37.82; P < 0.001/adjusted P = 0.07; and OR = 0.11; 95% CI, 0.03-0.35; P < 0.001/adjusted P = 0.06, respectively). Five WGCNA modules and 9 classes were associated with risk overall at FDR ≤ 0.20. Triacylglycerols (TAG) showed heterogeneity by tumor aggressiveness (case-only heterogeneity P < 0.0001). The TAG association with risk overall and serous tumors differed by acyl carbon content and saturation. In summary, this study suggests that pseudouridine may be a novel risk factor for ovarian cancer and that TAGs may also be important, particularly for rapidly fatal tumors, with associations differing by structural features. SIGNIFICANCE: Pseudouridine represents a potential novel risk factor for ovarian cancer and triglycerides may be important particularly in rapidly fatal ovarian tumors.

Plasma pseudouridine levels reflect body size in children on hemodialysis.

BACKGROUND: Dialysis in children as well as adults is prescribed to achieve a target spKt/Vurea, where Vurea is the volume of distribution of urea. Waste solute production may however be more closely correlated with body surface area (BSA) than Vurea which rises in proportion with body weight. Plasma levels of waste solutes may thus be higher in smaller patients when targeting spKt/Vurea since they have higher BSA relative to body weight. This study measured levels of pseudouridine (PU), a novel marker solute whose production is closely proportional to BSA, to test whether prescription of dialysis to a target spKt/Vurea results in higher plasma levels of PU in smaller children. METHODS: PU and urea nitrogen (ureaN) were measured in plasma and dialysate at the midweek hemodialysis session in 20 pediatric patients, with BSA ranging from 0.65-1.87m2. Mathematical modeling was employed to estimate solute production rates and average plasma solute levels. RESULTS: The dialytic clearance (Kd) of PU was proportional to that of ureaN (average KdPU/KdUreaN 0.69 ± 0.13, r2 0.84, p < 0.001). Production of PU rose in proportion with BSA (r2 0.57, p < 0.001). The pretreatment plasma level of PU was significantly higher in smaller children (r2 0.20, p = 0.051) while the pretreatment level of ureaN did not vary with size. CONCLUSIONS: Prescribing dialysis based on urea kinetics may leave uremic solutes at higher levels in small children. Measurement of a solute produced proportional to BSA may provide a better index of dialysis adequacy than measurement of urea.

Plasma metabolites and lipids associate with kidney function and kidney volume in hypertensive ADPKD patients early in the disease course.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity. METHODS: To investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey's false discovery rate (FDR) q-values to correct for multiple testing. RESULTS: Twelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value < 0.05. Specific significant metabolites, including pseudo-uridine, indole-3-lactate, uric acid, isothreonic acid, and creatinine, have been previously shown to accumulate in plasma and/or urine in both diabetic and cystic renal diseases with advanced renal insufficiency. CONCLUSIONS: This study identifies metabolic derangements in early ADPKD which may be prognostic for ADPKD disease progression. CLINICAL TRIAL: HALT Progression of Polycystic Kidney Disease (HALT PKD) Study A; Clinical www.clinicaltrials.gov identifier: NCT00283686; first posted January 30, 2006, last update posted March 19, 2015.

Validation of a Metabolite Panel for a More Accurate Estimation of Glomerular Filtration Rate Using Quantitative LC-MS/MS.

BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.

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.

Predictive metabolomics evaluation of nutrition-modulated metabolic stress responses in human blood serum during the early recovery phase of strenuous physical exercise.

We have investigated whether postexercise ingestion of carbohydrates in combination with proteins generates a different systemic metabolic response, as compared to the sole ingestion of carbohydrate or water, in the early recovery phase following exercise. In addition, metabolic patterns related to fitness level were studied together with individual responses to nutritional modulation. Twenty-four male subjects were exposed to 90 min of ergometer-cycling. Each participant was subject to four identical test-sessions, including ingestion of one of four beverages (water, low-carbohydrate beverage, high-carbohydrate beverage, and low-carbohydrate-protein beverage (LCHO-P)) immediately after cycling. Blood was collected at six time points, one pre- and five postexercise. Extracted blood serum was subject to metabolomic characterization by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS). Data was processed using hierarchical multivariate curve resolution (HMCR), and multivariate statistical analysis was carried out using orthogonal partial least-squares (OPLS). Predictive metabolomics, including predictive HMCR and OPLS classification, was applied to ensure efficient sample processing and validation of detected metabolic patterns. Separation of subjects in relation to ingested beverage was detected and interpreted. Pseudouridine was suggested as a novel marker for pro-anabolic effect following LCHO-P ingestion, which was supported by the detected decrease of the catabolic marker 3-methylhistidine. Separation of subjects according to fitness level was achieved, and nutritional modulation by LCHO-P was shown to improve the metabolic status of less fit subjects in the recovery phase. In addition, the potential of the methodology for detection of early signs of insulin resistance was also demonstrated.

Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection.

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.

Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.

We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.

[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.

[Determination of pseudouridine in serum by high performance liquid chromatography].

Pseudouridine is a modified nucleoside derived from the degradation of transfer ribonucleic acid. The elevation of modified nucleosides in urine has been suggested to be caused by higher turnover rate of t-RNA in tumor tissue than in healthy tissue, rather than by cell death. A method of pseudouridine determination in serum was developed by high-performance liquid chromatography on a Nova-Pak column (Waters) with 0.04 mol/L KH2PO4 (pH 4.0) as mobile phase. The blood samples were collected and 0.6 mL of serum was treated with 0.4 mL 6% HClO4. The precipitate was centrifuged for 10 min at 3000 r/min. Five-hundred microL of the liquor was dried by air-stream at 60 degrees C. The residue was dissolved with 300 microL mobile phase and 10 microL was injected. The average recovery was 93.50 +/- 2.1%. The calibration curve was linear within the concentration range of 0.7-6.8 micromol/L. The serum pseudouridine concentrations for patients with hepatitis, lung cancer, nephritis and uremia were determined and those of patients with lung cancer and uremia were found significantly higher than those of healthy controls (p<0.05). And for patients treated with He-Ne laser no significant change of the pseudouridine hasn't been found (p<0.05).

Ascitic pseudouridine discriminates between hepatocarcinoma-derived ascites and cirrhotic ascites.

Various biochemical indexes discriminate neoplastic from nonneoplastic ascites. However, within the latter group, the distinction between cirrhotic ascites and ascites caused by hepatocarcinoma (HC) is usually based on liver biopsy or cytology. HC-derived ascites is included in the group of nonneoplastic ascites because it is not associated with peritoneal spreading of neoplastic cells. In 54 cases of cirrhotic ascites and 17 cases of HC ascites, all histologically diagnosed, ascitic pseudouridine concentrations discriminated cirrhotic from HC ascites. For example, using the cutoff value of 4.25 mumol/L (obtained by ROC curve analysis) resulted in a diagnostic sensitivity of 88.2% and a diagnostic specificity of 90.8%. Moreover, in cirrhosis, the ascitic concentrations of pseudouridine were lower than serum concentrations, and the two sets of values were correlated; in HC, however, ascitic pseudouridine concentrations were higher than serum concentrations, and the two were unrelated. These findings strongly suggest that in cirrhotic patients ascitic pseudouridine derives from serum by diffusion, whereas in HC patients the mechanism appears to be more complex.

Pseudouridine and 1-ribosylpyridin-4-one-3-carboxamide (PCNR) serum concentrations in human immunodeficiency virus type 1-infected patients are independent predictors for AIDS progression.

A prospective study was conducted with 161 human immunodeficiency virus (HIV)-positive patients to investigate the prognostic role of 10 serum-modified nucleosides with regard to some of the most widely used parameters of AIDS progression. Serum concentrations of pseudouridine (> 3.77 nmol/mL) predicted progression to AIDS in CDC stage A2 HIV-infected patients much better than did other widely used parameters (hazard ratio, 2.7; 95% confidence interval, 1.29-6.35; P = .01; median permanence time in stage A2, 17 vs. 30.5 months; P = .03). Serum concentrations of 1-ribosylpyridin-4-one-3-carboxamide (PCNR) and beta 2-microglobulin and the CD4:CD8 cell ratio, in decreasing order and used in combination, differentiated the overall survival time probability of AIDS patients; PCNR was the best and a new independent predictor (overall survival time, > 31 months, no positive parameters; 19.3 months, one positive parameter; and 5.5 months, two positive parameters.

[Serum 1-methyladenosine and pseudouridine as tumor markers in tumor-bearing mice].

It is known that in cancer patients elevated levels of modified nucleosides originated from RNA are excreted in the urine. Modified nucleosides in the serum are thought to be more useful than those in the urine as tumor markers because they are not influenced by other factors. However the determination of these nucleosides is difficult because of their low amounts. To examine the efficacy of the modified nucleosides in the serum as tumor markers, ascites and solid tumor mouse models were prepared, and the amounts of 1-methyladenosine and pseudouridine in the serum were determined. Along with the growth of ascites tumor, the amounts of 1-methyladenosine and pseudouridine in the serum increased. The modified nucleosides in the serum in a solid tumor model also increased. This is the first report on the variation in the amount of 1-methyladenosine in the serum of tumor models, and the results suggest the usefulness of measuring the amounts of 1-methyladenosine and pseudouridine in the serum as tumor markers.

Capillary electrophoresis as a clinical tool. Determination of organic anions in normal and uremic serum using photodiode-array detection.

We report the use of free solution capillary electrophoresis to identify and quantify low-molecular-mass compounds found in normal and uremic serum as well as in hemodialysate fluid. The method reported provides a multicomponent analysis, allowing a single-step screening for more than 19 metabolites in less than 16 min. Serum samples from healthy individuals and from patients who have been diagnosed with chronic renal failure are analyzed using a borate buffer system at pH 9.0, and an extended light path capillary. Several ionic sample constituents are identified by electrophoretic mobility, UV spectra, and spiking with authentic standards. An analysis of the relative concentration of several metabolites, including hypoxanthine, pseudouridine, hippuric acid, and uric acid is presented. Each of these four metabolites is found in both normal and uremic serum samples (limits of detection 1 to 6 microM). Moreover, each of these metabolites is present at significantly elevated levels in uremic patients. The method reported is shown to have promising clinical utility for profiling serum sample constituents, and for quantitative determination of a few important metabolites.

Clinical value of urinary and serum pseudouridine in diagnosis and monitoring of primary liver cancer.

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.

Serum pseudouridine in the diagnosis of acute leukaemias and as a novel prognostic indicator in acute lymphoblastic leukaemia.

The serum level of pseudouridine, a modified nucleoside deriving mainly from t-RNA catabolism, was evaluated in 66 acute leukaemia patients at diagnosis to investigate its diagnostic and prognostic value, and its potential as a parameter with which to classify subtypes of the disease. Serum pseudouridine, measured by high performance liquid chromatography, was increased in acute lymphoblastic leukaemia patients (90% according to the pseudouridine index, which is the serum pseudouridine/creatinine ratio), and in acute myeloblastic leukaemia patients (75% according to the pseudouridine index). The increase was higher in the L3 than in the L1 and L2 subtypes. In the acute lymphoblastic leukaemia group there was a highly significant inverse correlation between serum pseudouridine levels and the most common end-point parameters used to assess disease outcome in leukaemia (i.e., complete remission rate, disease-free survival, and overall survival). In addition, 83% of patients with serum pseudouridine values < 5.5 nmol/mL were alive and in complete remission 12 months after the initial diagnosis, while only 11% of patients with serum pseudouridine values > 5.5 nmol/mL were alive and none were disease-free after the same period. This study: 1. demonstrates that the diagnostic sensitivity of the pseudouridine index is high in adult acute lymphoblastic leukaemia and good in acute myeloblastic leukaemia; 2. suggests that the serum pseudouridine assay can contribute to the classification of adult acute lymphoblastic leukaemia; and 3. demonstrates unequivocally that both pseudouridine assay and the pseudouridine index are excellent independent prognostic markers for acute lymphoblastic leukaemia.

Blood plasma pseudouridine in patients with malignant proliferative diseases.

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)

Efficiency of hemodialysis of pyrimidine compounds in patients with chronic renal failure.

The accumulation in blood plasma and efficiency of hemodialysis of pyrimidine compounds (orotic acid, orotidine, pseudouridine, uridine, thymine) as well as uric acid and creatinine in 23 patients with chronic renal failure (CRF) was investigated. As a reference, the analysis of the above metabolites in the plasma of 30 healthy volunteers was performed. Among examined compounds, pseudouridine possessed the highest capability of accumulation in blood plasma (25 times higher concentration than physiological). It coincided with the lowest efficiency of pseudouridine hemodialysis (44%) and the longest T1/2 (relative to creatinine) in plasma. A significant linear correlation (r = 0.81, p < 0.001) between efficiency of creatinine and pseudouridine hemodialysis was calculated. The concentration of orotic acid in the blood plasma of patients before hemodialysis exceeded 14 times its level in healthy subjects; the inhibition of uric acid synthesis by allopurinol in dialyzed patients was accompanied by enlargement of orotidine and orotate accumulation in blood plasma. Extremely high plasma concentration of examined pyrimidines remaining elevated after hemodialysis creates an additional hazard for tissue metabolism and health of patients with CRF.