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.

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 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.

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.

[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.

[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.

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.

Serum pseudouridine as a biochemical marker in patients with hepatocellular carcinoma.

The concentration of serum pseudouridine, a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in patients with hepatocellular carcinoma, liver cirrhosis, other benign hepatobiliary diseases, and healthy controls. The serum pseudouridine concentration in patients with hepatocellular carcinoma was significantly higher than that in patients with cirrhosis or the controls. Twenty-seven (51.9%) of 52 patients with hepatocellular carcinoma had serum pseudouridine concentrations higher than the mean value for healthy controls plus 2 SD. Fourteen of the 36 patients who had serum alpha-fetoprotein levels below 400 ng/ml, had elevated serum pseudouridine concentration. In total, 36 of the 52 patients (69.2%) could be detected by combination of these two markers. Two patients who had developed hepatocellular carcinoma during the course of cirrhosis and were continuously negative for alpha-fetoprotein, had higher levels of the pseudouridine concentration when hepatocellular carcinoma occurred. Furthermore, 4 of the 7 patients who had a very small cancer and were negative for alpha-fetoprotein, had elevated serum pseudouridine concentration. These results indicate that serum pseudouridine is a useful biochemical marker and that serum pseudouridine and alpha-fetoprotein in combination are considered to serve as complementary markers, for the diagnosis of hepatocellular carcinoma.

The use of receiver operating characteristic (ROC) curves analysis in the evaluation of the diagnostic efficiency of serum pseudouridine as a tumor marker.

The biochemical parameters used in this study were: (1) serum pseudouridine, expressed as nmols/mL; (2) pseudouridine index, expressed as mol to mol ratio of serum pseudouridine versus serum creatinine concentration. The receiver operating characteristic (ROC) analysis has been used to exemplify the selection of discriminant values or "cut-off points" to maximize the diagnostic utility of a biochemical tumor marker, serum pseudouridine. This marker has been used in a variety of group population samples, i.e., normal subjects, subjects affected by several nonneoplastic diseases, subjects with neoplastic disorders in less advanced or more advanced stages, and finally in a sample population of patients affected by lymphomas and leukemias of different types. An analysis of the relative ROC curves allowed the selection of cut-off values that maximize the diagnostic efficiency or, alternatively, the diagnostic sensitivity or the diagnostic specificity for pseudouridine parameters, and has allowed the comparison of the two tests to answer the same clinical question.

Serum pseudouridine as a biochemical marker in small cell lung cancer.

The serum level of pseudouridine, primarily a degradation product of tRNA, was determined by high-performance liquid chromatography in 24 patients with small cell lung cancer (SCLC), 13 patients with non-SCLC with advanced stages, 15 patients with pulmonary infectious diseases, and 18 healthy controls. The mean serum pseudouridine concentration was significantly higher in the patients with SCLC [4.75 +/- 1.76 (SD) nmol/ml] than that in the patients with pulmonary infectious diseases (3.39 +/- 1.38 nmol/ml) or in healthy controls (2.21 +/- 0.78 nmol/ml). The mean serum pseudouridine concentration in the patients with non-SCLC (4.07 +/- 0.95 nmol/ml) was significantly higher than that in healthy controls but not statistically different from that in the patients with pulmonary infectious diseases. The serum pseudouridine level was elevated above the mean value plus 2 SD for the healthy subjects (3.77 nmol/ml) in 66.7% of all patients with SCLC including 3 of 8 (37.5%) with limited disease and 13 of 16 (81.3%) with extensive disease, and 53.8% of the patients with non-SCLC. Serum carcinoembryonic antigen was elevated (greater than 5 ng/ml) in 29.2% and serum neuron-specific enolase (greater than 10 ng/ml) in 58.3% of the cases with SCLC. In the patients with SCLC followed up during chemotherapy, serum pseudouridine levels changed considerably parallel with the changes in the clinical response. These findings indicate that serum pseudouridine may be a useful biochemical marker in the patients with SCLC.

[Combination of cancer markers in the detection and prognosis of digestive system tumors]. / Combinação de marcadores de câncer na detecção e prognóstico de tumores do aparelho digestivo.

The role of carcinoembryonic antigen (CEA), gamma glutamyl transpeptidase (gamma GT), phosphohexose isomerase (PHI), pseudouridine (PSD) and acute phase reactant proteins (C-reactive protein (C-RP], alpha 1-acid glycoprotein (alpha 1-AGP) and alpha 1-antichymotrypsin (alpha 1-ACT) in distinguishing benign from malignant conditions of the digestive tract and their value in assessing the prognosis of gastrointestinal neoplasms was evaluated. For each marker it has been possible to produce a simple scoring index derived from the values found in the control population. This work suggests that pre-operative levels of certain cancer markers may be useful in tumor detection and may also give prognostic information additional to pathological staging.

Serum pseudouridine as a biochemical marker in the development of AKR mouse lymphoma.

Pseudouridine is a modified nucleoside derived from the degradation of some species of RNA, primarily transfer RNA, the level of which is elevated in biological fluids of tumor-bearing subjects. In order to study the relationship between pseudouridine levels and the development and progression of neoplasia, we have measured pseudouridine levels in the serum of inbred mice with high (AKR) and low (BALB/c) incidence of spontaneous lymphoma and in mice carrying transplantable lymphoid tumors. Our results show that the serum level of pseudouridine: (a) in healthy mice, is higher in females than in males; (b) increases significantly in female AKR mice in the period preceding the development of lymphoma (preneoplastic period occurring at about 6 months of age); and (c) is highest in AKR mice with lymphoma, the most elevated levels being found in mice with widely disseminated disease. The latter observation was confirmed by experiments with a transplantable AKR lymphoma (T2), where a positive correlation between tumor burden and serum pseudouridine levels was found. On the contrary, in BALB/c mice carrying a transplantable myeloma tumor (MOPC-460), no increase was seen despite the presence of a considerable tumor burden. The increase of pseudouridine in the preneoplastic period, in the absence of overt disease is viewed as an early sign of the development of the disease.

Determination of pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography.

A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reverse-phase high-performance liquid chromatography. The pseudouridine mean value in 30 normal subjects was 2.52 +/- 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios (A280/A254) with those of pure compounds, as well as by cochromatography.

Pseudouridine determination in blood serum as tumor marker.

A new method to measure pseudouridine (psi rd) in blood serum has been used to study patients with cancer of breast, lung, colon, and ovaries, and patients with hepatomas, non-Hodgkin lymphomas, and acute lymphoblastic leukemias, at different stages. The results indicate: (1) a significant (p less than 0.01) difference between the psi rd serum level of the cancer patient group and the normal control group; (2) that the increase of the level of serum psi rd is correlated both to the stage of the disease and to the spreading of the tumor tissue; (3) a correlation between the response to therapy and the behaviour of psi rd serum levels.