Pattern recognition of 8-hydroxy-2'-deoxyguanosine in biological fluids.

8-Hydroxy-2'-deoxyguanosine (8-OHdG), a product of oxidative DNA damage, which has been used as a sensitive and reliable marker of oxidative stress and carcinogenesis, is found in high levels in biological fluids of leukemia patients. A reliable screening method based on pattern recognition of 8-OHdG using stochastic sensors designed with graphene materials decorated with nanoparticles of TiO2Ag or TiO2Au was developed. 5,10,15,20-Tetraphenyl-21H,23H porphyrin (P), 2,6-bis((E)-2-(furan-2-yl)vinyl)-4-(4,6,8-trimethylazulen-1-yl)pyridine (Py1), and 2,6-bis((E)-2-(thiophen-3-yl)vinyl)-4-(4,6,8-trimethylazulen-1-yl)pyridine (Py2) were used as modifiers of the graphene paste in the design of sensors. The screening method used for pattern recognition was developed for two pH values accordingly with the nature of the biological fluid to be screened: pH = 3.02 for urine samples and pH = 7.49 for whole blood samples. High sensitivities and low limits of determination for 8-OHdG obtained at both pH values favored the early detection of leukemia. Recoveries over 98.00% with RSD (%) values lower than 1.00% proved the reliability of the proposed screening method. Graphical abstract Graphene based sensors detect 8-hydroxy-2'-deoxyguanosine in biological fluids.

Yield of Urinalysis Screening in Pediatric Cancer Survivors.

BACKGROUND: The Children's Oncology Group (COG) publishes consensus guidelines with screening recommendations for early identification of treatment-related morbidities among childhood cancer survivors. We sought to estimate the yield of recommended yearly urinalysis screening for genitourinary complications as per Version 3.0 of the COG Long-Term Follow-Up Guidelines and identify possible risk factors for abnormal screening in a survivor population. PROCEDURE: A database of pediatric cancer survivors evaluated between January 2008 and March 2012 at Children's Healthcare of Atlanta was queried for survivors at risk for genitourinary late effects. The frequency of abnormal urinalyses (protein ≥1+ and/or presence of glucose and/or ≥5 red blood cells per high power field) was estimated. Risk factors associated with abnormal screening were identified. RESULTS: Chart review identified 773 survivors (57% male; 67% Caucasian; 60% leukemia/lymphoma survivors; mean age at diagnosis, 5.7 years [range: birth to 17.7 years]; time from diagnosis to initial screening, 7.6 years [range: 2.3 to 21.5 years]) who underwent urinalysis. Abnormal results were found in 78 (5.3%) of 1,484 total urinalyses. Multivariable analysis revealed higher dose ifosfamide (odds ratio [OR] = 6.8, 95% confidence interval [CI] 2.9-16.0) and total body irradiation (TBI, OR = 3.0, 95% CI 1.0-8.4) as significant risk factors for abnormal initial urinalysis screening. CONCLUSIONS: Pediatric cancer survivors exposed to higher dose ifosfamide or TBI may be at higher risk of abnormal findings on urinalysis screening. Targeted screening of these higher risk patients should be considered.

Evaluation of the oxidative deoxyribonucleic acid damage biomarker 8-hydroxy-2'-deoxyguanosine in the urine of leukemic children by micellar electrokinetic capillary chromatography.

Determining the level of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, is vital to the study of clinical pathogenesis and drug toxicity. The principal limitation of capillary electrophoresis (CE) with UV detection is its low sensitivity. To overcome this shortcoming, we developed a micellar electrokinetic capillary chromatography (MEKC) with solid-phase extraction (SPE) for urinary 8-OHdG analysis. The sensitivity of MEKC-UV was improved using a reasonable UV system, injection mode, and SPE. The parameters affecting MEKC and SPE were also optimized. The calibration curve was linear within the range from 1 to 500 µg L(-1). The limits of detection and quantification were 0.27 µg L(-1) and 0.82 µg L(-1), respectively. Interday and intraday precision were both <5.6%. The recovery of 8-OHdG in urine ranged from 94.5% to 103.2%. This method was used to measure urinary 8-OHdG from eight normal children, eight newly diagnosed leukemic children, and eight leukemic children undergoing chemotherapy. The results show that the proposed method can be used to assess oxidative stress in patients and the side effects of chemotherapeutic drugs by measuring urinary 8-OHdG.

Urinary cytokines after HCT: evidence for renal inflammation in the pathogenesis of proteinuria and kidney disease.

We compared urinary levels of cytokines in patients with and without albuminuria, proteinuria and kidney disease (glomerular filtration rate<60 mL/min per 1.73 m(2)) after HCT. Plasma and urine were collected at baseline and weekly through day 100 and monthly through year 1, for measurement of IL-6, gp130, sIL6r, IL-10, IL15, MCP-1 and urine albumin-to-creatinine ratios (ACRs). Cox-proportional hazards modeling examined associations between urinary cytokine levels and development of these renal end points. The association of ACR with the hazard of overall mortality was assessed using Cox regression. Increasing urinary IL-6 and IL-15 were associated with an increased risk of developing proteinuria. Urinary MCP-1 during the first 100 days post HCT was associated with kidney disease at 1 year. The degree of albuminuria at any time point in the first 100 days post transplant was related to the subsequent risk of death (for ACR 30-299, hazard ratio (HR)=1.91; 95% confidence interval (CI): 1.27-2.87; for ACR >300, HR=2.82; 95% CI: 1.60-4.98). After HCT, elevated urinary levels of pro-inflammatory cytokines are associated with development of albuminuria and proteinuria, suggesting early intra-renal inflammation as an important pathogenetic mechanism. Albuminuria and proteinuria within the first 100 days post HCT are associated with decreased overall survival.

The impact of saturable metabolism on exposure-response relations in 2 studies of benzene-induced leukemia.

Enzymatic saturation of metabolic pathways is one factor that potentially contributes to the nonlinear exposure-response relations that are frequently reported in occupational epidemiologic studies. The authors propose an approach to explore the contribution of saturable metabolism to previously reported exposure-response relations by integrating predictive models of relevant biomarkers of exposure into the epidemiologic analysis. The approach is demonstrated with 2 studies of leukemia in benzene-exposed workers, one conducted in the Australian petroleum industry (1981-1999) and one conducted in a US rubber hydrochloride production factory in Ohio (1940-1996). The studies differed greatly in their magnitudes and durations of exposure. Substitution of biomarker levels for external estimates of benzene exposure reduced the fold difference of the log relative risk of leukemia per unit of cumulative exposure between the 2 studies by 11%-44%. Nevertheless, a considerable difference in the log relative risk per unit of cumulative exposure remained between the 2 studies, suggesting that exposure misclassification, differences in study design, and potential confounding factors also contributed to the heterogeneity in risk estimates.

[State of bone structures in children with acute leukemia, experiencing unfavorable factors from Chernobyl accident].

The authors present an assessment of bone structure condition in children with acute leukemia. The changes in the collagen molecule, amino acid composition of urine and proteins reparation processes were revealed. Calcium phosphate excretion in the patients urine were increased. The serum osteocalcin level and colony formation efficiency of bone marrow fibroblasts in acute leukemia patients are lower than in control group. In the initial period of the disease 32% of patients have disturbancies in their endocrine status. The bone structure violation is combined with unfavorable disease outcome. The effectiveness of the treatment and prevention steps in acute leukemia patients depends on the leukemic process stage.

Role of GSTM1, GSTP1, and GSTT1 gene polymorphism in ifosfamide metabolism affecting neurotoxicity and nephrotoxicity in children.

The aim of this study was to evaluate the impact of GSTM1, GSTT1, and GSTP1 gene polymorphism on urinary excretion of unchanged ifosfamide, 2-dechloroethylifosfamide (2DCIF), and 3-dechloroethylifosfamide (3DCIF) with regard to the incidence of ifosfamide-related nephrotoxicity and neurotoxicity in children. The study comprised 76 children (38 girls, 38 boys) ages 9.84 to 210 months who were being treated for various malignant diseases with ifosfamide. The children were enrolled after identification of genotype coding for three classes of glutathione S-transferases (GSTM1, GSTT1, and GSTP1) at the initial stage of diagnosis. (P) nuclear magnetic resonance spectroscopy was used to analyze the urinary excretion of unchanged ifosfamide, 2DCIF, and 3DCIF metabolites on consecutive days after the end of the 3-hour infusion of ifosfamide. In children with polymorphic locus of the GSTP1 gene compared with children with homozygous wild alleles, increased urinary excretion of 3DCIF (P=0.029) and decreased creatinine clearance was found (Mann-Whitney P=0.03; median 81.1 mL/min/1.73 m vs. 105.0 mL/min/1.73 m, respectively). The authors' multidimensional analysis model revealed that besides the total ifosfamide dose and co-administration of other toxic drugs, polymorphic locus of GSTP1 gene may be one of the factors determining a higher toxicity of the cytostatic agent. The model was construed at P=0.029. Moreover, no correlation was found between the GSTM1 or GSTT1 genotype and ifosfamide toxicity and the urinary excretion of its metabolites. The results of this analysis indicate that individual reactions to ifosfamide can depend on inherited genetic polymorphisms, especially associated with the GSTP1 gene coding detoxifying enzyme.

Elevated urinary dopamine in adults and children.

BACKGROUND: Information on the significance of an elevated urinary dopamine is limited and can lead to misinterpretation of the cause of such a finding. This laboratory-based study examines the associations with elevated dopamine gathered from a significant number of patients. METHODS: The urine catecholamine and metabolite results of specimens (analysed by HPLC-ECD) from 5933 adults and 467 children were examined retrospectively over a 57-month period. Those with elevated dopamine were identified and the explanation for this finding was sought. RESULTS: In adults, the conditions associated with an elevated dopamine were: urine over-collection; drug effects (including those due to intravenous dopamine, L-dopa, methyldopa, clozapine, antidepressants and metoclopramide); clinical effects (including those due to phaeochromocytoma, carcinoid tumour and pregnancy). In children, high urine dopamine was found in cases of neuroblastoma, Costello syndrome, leukaemia, phaeochromocytoma, Menkes disease and rhabdomyosarcoma of the bladder. CONCLUSIONS: A high urine dopamine was found in <3% of adult urine specimens. It was most commonly associated with: over-collection, probable drug effects and neural crest tumours. Neuroblastoma was the most common cause of elevated dopamine in children's specimens, although other associations are described. Some await explanation.

Urinary excretion of beta-aminoisobutyric acid in hematological diseases.

The level of beta-aminoisobutyric acid (beta-AIB), a thymine catabolite, has been measured in urine samples of 160 healthy individuals, 28 patients with renal, 27 patients with cardiovascular and 27 patients with hematological diseases and of 36 tumor patients. No significant difference in the prevalence of high excretors of beta-AIB between patients with cancer, renal and cardiovascular diseases and the healthy group was found, whereas all but two patients with hematological diseases were high excretors. Urinary beta-AIB shows a reverse correlation with the hemoglobin level and erythrocyte count in the cases of anemia, and appears to be directly correlated with the leukocyte count and blast cell content in the cases of leukemia, with its amount decreasing two to five-fold with the return of the hematological markers to normal levels after medicinal treatment. Therefore the beta-AIB concentration in urine may be used in combination with hematological indicators in assessing the disease status and in monitoring of the treatment response.

Urinary excretion of N-acetyl-beta-D-glucosaminidase and alpha1-microglobulin in children with proliferative blood diseases.

The purpose of the study was to estimate the urinary excretion of NAG and alpha-1M among children who suffer from proliferative blood diseases. The group of the examined children included those who went through a viral hepatitis (VH) and who are or were treated by means of cytostatic drugs. The study comprised 73 children aged from 4 to 18 (average 11.7+/-3.5. There were 70 children with the diagnosis of leukemia and 3 with the diagnosis of non-Hodgkin lymphoma. The examined group was divided according to the stage of treatment of a basic disease. Group I--22 children who are treated currently or whose treatment has been completed recently. Group II--51 children whose treatment was completed over two years ago. In group II there were 4 subgroups distinguished depending on positive antigenemia HBs and the presence of HCV antibodies. There were no clinical or biochemical features of damage of renal function observed among any of the children. The testing group consisted of 70 healthy children who were selected regarding age and sex. The urinary excretion of NAG and alpha-1M was estimated in the second morning portion of urine and it was presented as NAG/creatinine and alpha-1M/creatinine ratio. The results of the research underwent the statistical analysis by means of a t-Student test. It was stated that the urinary excretion of NAG and alpha-1M was higher among children who currently are or were treated by means of cytostatics drugs. It was also stated that the urinary excretion of NAG was higher among the children who went through viral hepatitis C in comparison with HBs antigen carriers. Similarly, the urinary excretion of alpha-1M was higher among children with positive markers of viral hepatitis B and C markers in comparison with a group of HBs antigen carriers.

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.

Dihydropteridine reductase activity and neopterin levels in leukemias and lymphomas: is there any correlation between these two parameters?

Urinary neopterin levels, blood dihydropteridine reductase activity as well as other frequently used clinical parameters were evaluated in 110 patients suffering from various types of lymphomas and leukemias. Among them neopterin was detected as the most sensitive marker representing the severity of malignancy (p<0.00001). All patients with active diseases had significantly raised urinary neopterin levels compared to those in remission and healthy controls. Of 69 patients with active disease 66 (96%) were above the upper limit seen in healthy subjects. In addition, the highest neopterin excretion was found in patients with active chronic myeloid leukemia (1469+/-479 micromol/mol creatinine n=16). In contrast, only 1 of 41 patients in stable responsive disease and remission (2.4%) had increased urinary neopterin levels above the upper limit. Dihydropteridine reductase (DHPR) activities were also detected in all patients and control groups. In active disease slightly reduced (DHPR) activities were evident (3.42+/-0.37 for controls, 2.92+/-0.39 in active disease and 3.28+/-0.42 nmol red cytochrome C/min/5 mm diameter disc in remission patients). However in patients under medication this was strengthened. This data also suggest that DHPR activity can be effected by chemotherapy. The results of the present study support the fact that urinary neopterin levels may be an useful and reliable early prognostic marker for neoplasia when used together with other prognostic indicators. Our data also suggest that reductions in DHPR activities may also be an underlying cause for the neurological disorders that are commonly seen in patients with haematological malignancies.

Two approaches for determining the urinary excretion patterns of nucleosides--HPLC and CE.

Cancer patients excrete in their urine increased amounts of modified purines and pyrimidines. These modified mucleosides are primary constituents of RNA and are synthesized at the macromolecular level. When RNA is catabolized, most of these modified nucleosides cannot be reutilized; consequently, they are excreted. Due to their increased urinary excretion in conjunction with altered RNA turnover in carcinogenesis, they have been proposed as tumour markers. We developed both reversed-phase high performance liquid chromatographic and capillary electrophoretic approaches to determine normal and modified nucleosides in urine. Data obtained with CE are in good accordance with those from HPLC. The reference excretion levels of urinary nucleosides from healthy volunteers were established, and elevated ones of modified nucleosides from 34 patients with different kinds of cancer were observed. The developed methods are suitable for the analysis of large series of samples for clinical studies.

Polyamine profiles in the urine of patients with leukemia.

Eleven urinary polyamine levels were determined in controls (32 cases) and 43 patients with varying stages of leukemia including initial, relapse and complete remission, using gas chromatography nitrogen-phosphorus detection. Also, to indirectly evaluate the possible involvement of enzymes, precursor to product concentration ratios were compared between controls and patients with each stage of leukemia. As a result, it is confirmed that the ratio of N1-acSpd/N8-acSpd could be used as a diagnostic marker and the level of N1,N12-diacetylspermine could be used for determining disease stage and as a malignancy marker for leukemia. An altered metabolic pathway related to leukemia is also proposed in which N1,N12-diacetylspermine can be produced directly from spermine and N1,N12-diacetylspermine is a major source of N1-acetylspermidine.

Urinary N-acetyl-beta-D-glucosaminidase activity in leukaemic children during high-dose methotrexate therapy.

Urinary N-acetyl-beta-D-glucosaminidase (NAG-ase) activity is a very sensitive parameter of kidney proximal tubular damage. Using urinary NAG-ase activity/urinary creatinine as the NAG index, the serum methotrexate (MTX) level and urinary pH were investigated simultaneously. These parameters were measured in 17 leukaemic children. During MTX treatment, NAG indices were normal in 5 children and only slightly elevated occasionally in 9 patients. Among them, transiently high serum MTX levels (Patient A) or low urinary pH (Patient B) were accompanied by high NAG indices. MTX toxicity has been diagnosed in 3 cases, when permanently high NAG indices were in accordance with other clinical signs.

Immunochemical detection of urinary 5-methyl-2'-deoxycytidine as a potential biologic marker for leukemia.

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2'-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2'-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2'-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2'-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 +/- 0.43 nmol/mumol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/mumol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2'-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2'-deoxycytidine might be applicable as a biologic marker for leukemia.

Comparison of urinary neopterin and pseudouridine in patients with malignant proliferative diseases.

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

Diagnostic use of anti-modified nucleoside monoclonal antibody.

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

Simultaneous determination of pseudouridine and creatinine in urine of normal children and patients with leukaemia by high performance liquid chromatography.

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