Urinary 5-hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.

CONTEXT: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer. OBJECTIVES: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-hmUra). MATERIALS AND METHODS: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited. RESULTS: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio). CONCLUSION: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.

A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS-UHPLC analysis of four urinary biomarkers.

In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS/UHPLC-PDA method were also very satisfactory. Finally, the application of the methodology to the determination of target biomarkers in normal subjects and CVDs patients' revealed that the DNA adducts 5-HMUra and 8-oxodG levels are much more abundant in CVDs patients while no statistic differences were obtain for MDA and UAc. This result points to the importance of 5-HMUra and 8-oxodG as biomarkers of CVDs risk progression and further epidemiological studies are needed to explore the importance of this correlation.

A micro-extraction technique using a new digitally controlled syringe combined with UHPLC for assessment of urinary biomarkers of oxidatively damaged DNA.

The formation of reactive oxygen species (ROS) within cells causes damage to biomolecules, including membrane lipids, DNA, proteins and sugars. An important type of oxidative damage is DNA base hydroxylation which leads to the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 5-hydroxymethyluracil (5-HMUra). Measurement of these biomarkers in urine is challenging, due to the low levels of the analytes and the matrix complexity. In order to simultaneously quantify 8-oxodG and 5-HMUra in human urine, a new, reliable and powerful strategy was optimised and validated. It is based on a semi-automatic microextraction by packed sorbent (MEPS) technique, using a new digitally controlled syringe (eVol(®)), to enhance the extraction efficiency of the target metabolites, followed by a fast and sensitive ultrahigh pressure liquid chromatography (UHPLC). The optimal methodological conditions involve loading of 250 µL urine sample (1:10 dilution) through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVol(®) syringe followed by elution using 90 µL of 20% methanol in 0.01% formic acid solution. The obtained extract is directly analysed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid and methanol in the isocratic elution mode (3.5 min total analysis time). The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), extraction yield, accuracy, precision and matrix effect. Satisfactory results were obtained in terms of linearity (r(2) > 0.991) within the established concentration range. The LOD varied from 0.00005 to 0.04 µg mL(-1) and the LOQ from 0.00023 to 0.13 µg mL(-1). The extraction yields were between 80.1 and 82.2 %, while inter-day precision (n = 3 days) varied between 4.9 and 7.7 % and intra-day precision between 1.0 and 8.3 %. This approach presents as main advantages the ability to easily collect and store urine samples for further processing and the high sensitivity, reproducibility, and robustness of eVol(®)MEPS combined with UHPLC analysis, thus retrieving a fast and reliable assessment of oxidatively damaged DNA.

Measurement of urinary excretion of 5-hydroxymethyluracil in human by GC/NICI/MS: correlation with cigarette smoking, urinary TBARS and etheno DNA adduct.

5-Hydroxymethyluracil (5-HMU) is derived from radiation in addition to endogenous oxidative DNA damage and it is one of the most abundant DNA adducts. Human 5-HMU-DNA-glycosylase has been shown to repair this lesion. Whether urinary levels of 5-HMU is a valid biomarker for oxidative DNA damage in vivo has been investigated. However, controversial results on its relation to cigarette smoking were reported. To facilitate analysis of urinary 5-HMU in epidemiological studies, a highly sensitive and specific assay based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry was developed. The limit of detection for N1,N3-bis(pentafluorobenzyl)-HMU is 10 fg (20 amol) (S/N=4) injected on column and the limit of quantification in urine was 0.7 nM of 5-HMU. Using as little as 10 microL of human urine samples, levels of urinary 5-HMU in 21 healthy volunteers were accurately quantified. No correlation was observed between urinary 5-HMU levels and cigarette smoking. However, there was a statistically significant association between urinary levels of 5-HMU and thiobarbituric acid-reactive substances (r=0.71, p=0.0003). In addition, urinary 5-HMU levels also correlated with urinary levels of 1,N6-ethenoadenine (r=0.54, p=0.01). These findings suggest that this assay should be valuable in assessing the role of urinary 5-HMU as a biomarker of oxidative DNA damage and repair.

Diet is not responsible for the presence of several oxidatively damaged DNA lesions in mouse urine.

In order to eliminate the possibility that diet may influence urinary oxidative DNA lesion levels, in our experiments we used a recently developed technique involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection. This methodology was applied for the determination of the lesions: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-(hydroxymethyl)uracil (5HMUra) in the urine of mice fed with nucleic acid free diet and normal, unrestricted diet. The mean levels of 8-oxoGua, 8-oxodGuo and 5HMUra of the animals fed the normal diet reached the mean values of 15.6 +/- 3.5, 2.0 +/- 0.53 and 16.8 +/- 10.4 nmol/kg/24 h, After feeding the mice for 12 days with nucleic acid free diet the respective values were 18.8 +/- 4.6, 1.6 +/- 0.3 and 25.4 +/- 10.5 nmol/kg/24 h, respectively. The results clearly demonstrate that irrespective of the diet, the excretion rates were not statistically different during the course of feeding. The respective p values for the differences between lesions in the two types of diets were: 0.13 (8-oxoGua), 0.16 (8-oxodGuo), 0.18 (5-HMUra). Our results clearly indicate that diet does not contribute to urinary excretion of the lesions in mouse model.

Simultaneous determination of five oxidative DNA lesions in human urine.

A method, involving a HPLC prepurification followed by a GC/MS analysis, has been set up for the measurement of nucleic acid oxidation products in human urine. For this purpose, isotopically labeled internal standards have been prepared and used for isotope dilution mass spectrometric detection. Using this approach, four oxidized DNA bases, i.e., 5-hydroxyuracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroadenine, and 8-oxo-7,8-dihydroguanine, together with 8-oxo-7,8-dihydro-2'-deoxyguanosine have been simultaneously quantified in human urine samples. The levels of the oxidized nucleic acid constituents, as expressed in picomoles per milliliter, were determined to be, in decreasing order: 8-oxo-7,8-dihydroguanine (583 +/- 376) > 5-(hydroxymethyl)uracil (121 +/- 56) > 5-hydroxyuracil (58 +/- 23) > 8-oxo-7,8-dihydro-2'-deoxyguanosine (30 +/- 15) > 8-oxo-7,8-dihydroadenine (7 +/- 4). Attempts to determine the amount of 5,6-dihydroxy-5,6-dihydrothymine, 5-hydroxycytosine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine using the above HPLC-GC/MS method were unsuccessful.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers.

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54+/-2.07 vs. 6.70+/-1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16+/-0.35 vs. 1.35+/-0.50 nmol/mmol creatinine, p = 0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r(p) = 0.44), BMI (r(p) = -0.27) and nicotine derivatives (r(p) = 0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r(p) = 0.36), nicotine derivatives (r(p) = 0.29), BMI (r(p) = -0.24) on the other.

Urine 8-oxo-7,8-dihydro-2-deoxyguanosine vs. 5-(hydroxymethyl) uracil as DNA oxidation marker in adriamycin-treated patients.

We measured the base 5-(hydroxymethyl) uracil (HMUra) and the nucleoside 8-oxo-7,8-dehydro-2'-deoxyguanosine (8-oxo-dGuo) in urine of adriamycin-treated cancer patients. Adriamycin has been shown to generate oxygen free radicals by various mechanisms. HMUra and 8-oxo-dGuo are two known lesions of DNA, produced by oxygen free reaction on thymine and 2'-deoxyguanosine, respectively. HMUra was measured by GC-MS/isotopic dilution and 8-oxo-dGuo by HPLC/EC, both after prepurification by semipreparative HPLC. Here we report the results of a study involving 20 cancer patients treated with flash doses of ADR. We found that urine HMUra is significantly increased (HMUra (nmol/24h): 80.8 8.44 vs. 98.7+/-6.87; p < 0.01) 24h after administration of the drug, while 8-oxo-dGuo did not show any significant variation. Urine HMUra seems to be a suitable short-term marker of DNA alterations by oxygen free radicals.

5-Hydroxymethyluracil excretion, plasma TBARS and plasma antioxidant vitamins in adriamycin-treated patients.

The thymine oxidative lesion-5-hydroxymethyluracil (HMUra)-was measured in urine collected from cancer patients. These patients all received chemotherapy using Adriamycin. Adriamycin (ADR) intercalates DNA coils and interferes with normal cell metabolism through diverse biochemical mechanisms that may explain its different actions. The anticancer action of ADR could derive from its interaction with topoisomerase II, resulting in DNA nicking followed by DNA fragmentation and apoptosis. Side effects of ADR-mainly its cardiotoxicity-may derive from the fact that ADR generates superoxide and hydroxyl radicals in two ways: redox-cycling and a Haber-Weiss type reaction due to Fe-ADR complexes. The oxygen free radicals, particularly .OH, are thought to be produced by ADR directly in genomic material and attack all its components. 5-Hydroxymethyluracil is a thymine lesion provoked by these attacks, and it has been proposed as a marker of DNA alterations. In this article, we report the results of a study involving 14 cancer patients treated with ADR. We found that urine HMUra is significantly increased by the anticancer therapy (HMUra (nmol/24 h): 74.4 9.46 vs. 96.3 8.74; p < .01), this increase reveals a higher risk of mutagenesis. Our study is the first to show an in vivo alteration of DNA by ADR. Results also show that thiobarbituric acid reactants increase significantly, and that the vitamin levels for retinol and alpha-tocopherol, which are antioxidant vitamins, are lower at the end of chemotherapy. We suggest to supplement these patients with vitamins A and E, and selenium to reduce the side effects of ADR.

Gas chromatographic-mass spectrometric determination of 5-hydroxymethyluracil in human urine by stable isotope dilution.

A method for the determination of 5-hydroxymethyluracil in urine is described. 5-Hydroxymethyluracil was extracted by reversed-phase chromatography and quantified by gas chromatography-mass spectrometry as tert.-butyldimethylsilyl derivative. Since natural 5-hydroxymethyluracil contained ca. 22% of M + 2 species, an internal standard consisting of [1,3-15N2,5-2H2]hydroxymethyluracil was used to correct losses during extraction, evaporation and derivatization. Between-run precision of this method was 7.79%, and concentrations as low as 1.87 nM could be measured. This sensitivity and precision could not be obtained with trimethylsilyl derivatives.

Dihydropyrimidine dehydrogenase deficiency leading to thymine-uraciluria. An inborn error of pyrimidine metabolism.

Three unrelated patients with excessive thymine-uraciluria due to dihydropyrimidine dehydrogenase deficiency are described. Excretory values (mmol/g creatinine) were: uracil 2.0-10.5, thymine 2.3-7.5, 5-hydroxymethyluracil 0.2-0.9. Orally administered (index patient) uracil and thymine were excreted for the greater part whilst dihydrouracil and S-dihydrothymine were mainly metabolised. Dihydropyrimidine dehydrogenase activities (nmol X h-1 X mg-1 protein) in leucocytes were 0.04, 0.01 and less than 0.01 in the patients, 0.31-1.66 in their parents, and 1.01-4.46 in controls (n = 4). The patients presented with a non-specific clinical picture of cerebral dysfunction.