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Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals
Journal of Photochemistry & Photobiology, B: Biology 153 (2015) 191–197
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jpb
Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals M. Zvarik a, D. Martinicky b, L. Hunakova c, L. Sikurova a,⁎ a b c
Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Computer Science, Comenius University, Bratislava, Slovakia Department of Gynecological Oncology, National Cancer Institute, Bratislava, Slovakia Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 1 April 2015 Received in revised form 17 August 2015 Accepted 16 September 2015 Available online 24 September 2015 Keywords: Fluorescence Pteridine Urine Ovarian tumors
a b s t r a c t Pteridines belong to a class of fluorescent metabolites that are excreted by humans in urine and their concentrations can reflect various pathophysiological states. We quantified the differences in urinary pteridine levels in patients with malignant and benign ovarian tumors and in healthy individuals. Urine samples were centrifuged and supernatants were oxidized by MnO2 before analysis. Levels of neopterin, biopterin, and pterin were assessed by fluorescence analysis of human urine after HPLC separation. We have revealed that the median neopterin levels were higher in urine samples from patients with malignant (0.226 μmol/mmol creatinine) and benign ovarian tumors (0.150 μmol/mmol creatinine) than in healthy subjects (0.056 μmol/mmol creatinine). The median neopterin levels of patients with malignant tumors were higher (1.5-times) than in patients with benign tumors. The median biopterin level in urine of patients with benign ovarian tumors (0.268 μmol/mmol creatinine) was found to be very close to the level in patients with malignant ovarian tumors (0.239 μmol/mmol creatinine), and both were higher than in healthy samples (0.096 μmol/mmol creatinine). The levels of urine pterin followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels. © 2015 Published by Elsevier B.V.
1. Introduction Pteridines are low molecular weight substances belonging to a class of fluorescent metabolites that are excreted by humans in urine. The naturally occurring pteridine derivatives exist in three different oxidation states: tetrahydro-, dihydro-, and fully-oxidized [1]. Fully oxidized pteridines are fluorescent compounds, while the fluorescence of reduced derivatives is much weaker [2,3]. Scientific data available to-date show that in humans, pteridines are synthesized from guanosine triphosphate (GTP) by GTPcyclohydrolase I [4]. GTP cyclohydrolase I converts GTP to 7,8dihydroneopterin-3′-triphosphate. This intermediate is further metabolized by 6-pyruvoyl tetrahydropterin synthase to 6-pyruvoyl tetrahydropterin, which is finally converted by two NADPHdependent reductions to tetrahydrobiopterin, the active hydroxylating compound. These NADPH-dependent hydroxylations are carried out by sepiapterin reductase [5].
⁎ Corresponding author at: Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Computer Science, Mlynska dolina F1, 84248 Bratislava, Slovakia. E-mail addresses: [email protected] (M. Zvarik), [email protected] (D. Martinicky), [email protected] (L. Hunakova), [email protected] (L. Sikurova).
http://dx.doi.org/10.1016/j.jphotobiol.2015.09.019 1011-1344/© 2015 Published by Elsevier B.V.
5,6,7,8-Tetrahydrobiopterin has been recognized as the most important unconjugated pteridine existing in biological human fluids [1]. Biopterin and dihydrobiopterin are the oxidative products of tetrahydrobiopterin [6]. In the human organism, tetrahydrobiopterin is involved in the hydroxylation reactions of phenylalanine, tyrosine and tryptophan. It is also an essential cofactor for the biosynthesis of the neurotransmitters dopamine, noradrenaline and serotonin, and of the reactive free radical nitric oxide. In addition, tetrahydrobiopterin also enters the metabolism of lipids as a cofactor of alkylglycerol monooxygenase (also called glyceryl ether monooxygenase, GEMO). GEMO is the only enzyme known to cleave the ether bond in alkylglycerol ether [7]. Tetrahydrobiopterin provides electrons being oxidized to dihydrobiopterin, which is recycled to tetrahydrobiopterin by action of the dihydropteridine reductase. Nearly all human cells like fibroblasts or endothelial cells produce tetrahydrobiopterin from GTP and only scarce amounts of neopterin species are formed. The only exception being monocytes/macrophages and monocyte derived dendritic cells, in which, due to the low activity of 6-pyruvoyl-tetrahydropterinsynthetase, relevant amounts of neopterin and dihydroneopterin are formed [5]. Several unconjugated pteridines, particularly neopterin, biopterin, xanthopterin, isoxanthopterin and pterin are responsible for blue autofluorescence of human urine [6]. Urinary excretion of these compounds has been found to increase as a result of several disorders such as viral
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infections, intracellular bacteria and parasites, chronic inflammatory disorders, or autoimmune diseases [8,9]. Using various fluorescence techniques, a crucial fact for diagnostics was established: the native blue fluorescence of urine pteridines in oncological patients is different from that in healthy individuals [10–12]. However, those data often came from a mixture of different tissues and tumor types [1,13,14]. Although Han et al. [14] stated that each type of tumor shows its own pattern in changes of pteridine concentrations in urine because different pteridine derivatives may play various roles in different tumor-related diseases, studies where the levels of pteridine derivatives in urine from patients with exclusively one type of tumor are assessed are scarce [15]. Ovarian cancer is the third most common gynecological cancer [16], but there is still lack of studies dealing with the quantification of pteridine levels in patients with ovarian malignancies [17–20], and urine pteridine studies focused on distinguishing between malignant and benign ovarian tumors are completely missing. The quantitative evaluation of pteridines is very important not only for a potential early diagnostics of the disease, but it also helps to explain the differences in metabolism of pteridines in various types of tumors and to tell apart malignant and benign tumors. In this study, we used high performance liquid chromatography (HPLC) analysis with fluorescence detection for quantitative evaluation of three pteridines (neopterin, biopterin, pterin) in urine of patients with ovarian tumors to establish differences between patients with malignant and benign ovarian tumors and healthy individuals. 2. Materials and Methods 2.1. Chemicals 6-Biopterin, D-erythro-neopterin, pterin, creatinine, methanol and water (HPLC grade) were purchased from Sigma-Aldrich Co. Sodium hydroxide, hydrochloric acid, monopotassium phosphate monobasic and manganese dioxide were obtained from Slavus Ltd. (Bratislava, Slovakia).
2.2. Buffer Preparation A 10 mmol/l aqueous solution of KH2PO4 sample buffer was prepared by diluting 10 ml of 1 mol/l KH2PO4 with water and by adding 30 ml of methanol. The buffer was filled with water to a final volume of 1 l and adjusted to pH 4.5 with HCl.
2.4. Urine Sample Preparation This study has been cleared by The National Cancer Institute (Slovakia) Ethics Review Board for human study and patients have signed an informed consent. 32 morning urine samples from fasting normal volunteers and 75 patients were used in this study. All of them were analyzed for pH, protein, glucose, bilirubin, nitrate, hemoglobin, ketones, acetone, and urobilinogen. The presence of red blood cells, white blood cells, casts, epithelial cells and crystals was also tested in these samples at the Department of Clinical Biochemistry, National Cancer Institute, Bratislava, Slovakia. Urine samples were taken from patients who did not undergo chemical or radiation therapy, they were taken before the start of anticancer or antibiotic therapy. The control group volunteers did not take any medications including vitamin supplements. The age distribution was 24–77 years for patients and 23–64 years for healthy subjects. The group of oncological patients was represented by 36 patients with confirmed ovarian malignant tumor (ICD 10 code C56) and 39 patients with ovarian benign tumor (ICD 10 code D27). Urine samples were centrifuged at 3000 rpm for 10 min at room temperature (22 ± 1 °C) and undiluted supernatants were used for analysis. All samples were stored in a freezer at −35 °C. Prior to analysis, the samples were removed from the freezer and brought to room temperature. Human urine samples were subjected to HPLC analysis after MnO2 oxidation. We pipetted 1 ml of fresh random urine into a centrifugation vial and adjusted pH to 1.0–1.5 with 40 μl of 6 mol/l HCl. After that we added 20 mg of MnO2 and shook for 5 min at room temperature. Then, we centrifuged for 5 min at 3000 ×g and immediately transferred clear supernatant into a vial. The vial was wrapped in aluminium foil to protect its content from light. 2.5. High Performance Liquid Chromatography System The chromatographic studies were performed on a modular HPLC system Prominence 20A (Shimadzu Co.), equipped with degasser (DGU-20A5), solvent delivery unit with quaternary pump (LC-20AD), autosampler with a sample cooler (SIL-20AC), column oven (CTO20AC), UV–VIS absorption detector (SPD-20A), fluorescence detector (RF-10AXL), system controller (CBM-20A) and the LC solutions software (1.25 SP1) to control the instrument, data acquisition and data analysis. The analytical columns used were Nucleosil® C18, 150 mm × 3.2 mm, 5 μm particle size (Supelco Analytical). 2.6. High Performance Liquid Chromatography Analysis
2.3. Standard Preparation Pteridine stock solutions (0.01% w/v) were prepared by dissolving separately 1 mg of neopterin, biopterin, or pterin in 1 ml 0.1 mol/l NaOH. We kept solutions in an ultrasound bath for 30 s, then we added 9 ml 0.1 mol/l HCl, and kept it in an ultrasound bath for another 30 s. Pteridine working solutions (0.001% w/v) were prepared by diluting 1 ml of each 0.01% pteridine stock solution with 9 ml 0.05 mol/l HCl. Urine standard mixture was prepared by pipetting 324 μl resp. 2530 μl (pterin resp. neopterin and biopterin) of 0.001% pteridine working solutions in a 20-ml flask and filling with 0.05 mol/l HCl to the mark. A dilution series of this mixture were used to generate calibration curves (1, 2, 3, 4 and 5 μmol/l for neopterin and biopterin, respectively; 0.2, 0.4, 0.6, 0.8 and 1 μmol/l for pterin). Standard creatinine stock solution was prepared by dissolving 45 mg creatinine in 10 ml 0.05 mol/l solution HCl to make the creatinine concentration of 40 mmol/l. This standard stock solution was diluted to an appropriate concentration with 0.05 mol/l solution of HCl (for calibration 5, 10, 20, 30 and 40 mmol/l). The standard solutions were stored at −35 °C.
Oxidized samples of urine were eluted isocratically with buffer solution (10 mmol/l aqueous solution of KH2PO4, 3% methanol) at a flow of 0.6 ml/min. The injected volume sample was 5 μl. After 20 min, when pteridines were eluted, the next sample could be injected. After 5 injections of biological samples, the columns were rinsed with aqueous solution of methanol (1:1) followed by buffer solution, to condition them for the next series of samples. The system temperature was adjusted to 25 °C. Detection was performed with photometric detector at 250 nm for creatinine analysis, and fluorimetric detector at 450 nm (excitation at 350 nm) for pteridine analysis, connected in serial.
2.7. Statistical Analysis Pteridine concentrations were determined by linear regression against experimentally generated calibration curves by LC solutions software (Shimadzu Co.). The collected data were graphically and statistically processed by the R software. The Mann–Whitney U test was used to compare medians of pteridine urine levels from healthy persons and patients.
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Fig. 1. Chromatogram of the creatinine standard (10 mmol/l). Absorption detection (250 nm).
3. Results Full oxidization of pteridines in the urine samples was selected for the reliability of pteridine fluorescence quantification. The levels of urine pteridines were normalized to urine creatinine concentrations so that fluctuations in urine concentration were minimized. The linearity of the HPLC method was evaluated by using standard creatinine solutions and standard mixtures of neopterin, biopterin, and pterin with different concentrations. The standard mixtures were injected into the HPLC by triplicate to obtain their elution order (Figs. 1 and 2). The acquired detection limit, linear equations and R2 values are given in Table 1. HPLC examination of human urine samples by UV absorption detection confirmed the narrow and intensive signal for creatinine (Fig. 3). Fluorescence chromatograms of all urine samples showed multiple peaks within a 15 min. time frame, where the peaks of neopterin and biopterin were convincingly identified and pterin provided weaker signal (Fig. 4). The levels of the all three pteridines studied in the urine from patients with malignant ovarian tumors were found to be significantly higher (p b 0.001) than those for the control group (healthy subjects) (Table 2). Moreover, the median value of urine neopterin concentration for cancer patients (0.226 μmol/mmol creatinine) exceeded the upper limit of healthy subjects (Fig. 5). The median levels of neopterin were less but significantly elevated (p b 0.001) also in the urine samples
from patients with benign ovarian tumor (0.150 μmol/mmol creatinine) as compared to controls (0.056 μmol/mmol creatinine). In addition, the level of urine neopterin was significantly elevated (p b 0.001) in patients with malignant ovarian tumors (0.226 μmol/mmol creatinine) as compared to those with benign ovarian tumors (0.150 μmol/mmol creatinine). Urine biopterin levels showed a different pattern in patients with benign ovarian tumor (0.268 μmol/mmol creatinine), acquiring nearly identical value to that found in samples from patients with malignant ovarian tumor (0.239 μmol/mmol creatinine); these values were significantly higher (p b 0.001) compared to healthy samples (0.096 μmol/mmol creatinine) (Fig. 6). Levels of urine pterin showed a pattern similar to neopterin, however pterin concentrations were about 3 times lower than those of neopterin and disease-related changes were observed with lower significance (Fig. 7). The median urine pterin levels for patients with malignant ovarian tumor (0.056 μmol/mmol creatinine) were significantly higher (p b 0.001) than the control level (0.020 μmol/mmol creatinine) and also than the level found in patients with benign ovarian tumors (0.041 μmol/mmol creatinine), but only with p b 0.05. The urine pterin level for patients with benign ovarian tumor was significantly higher (p b 0.01) than in healthy subjects. Spearman's rank correlation coefficients were found to be 0.715 for neopterin/ biopterin, 0.663 for neopterin/pterin, and 0.687 for biopterin/pterin.
Fig. 2. Chromatogram of the standard mixture of three pteridine standards. Fluorescence detection (excitation 350 nm, emission 450 nm).
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Table 1 Validation parameters of HPLC method. Compound
RT/min (RSD)
Area/mV·min (RSD)
R2
Slope
LOD (μmol/l)
LOQ (μmol/l)
Selectivity (α)
Creatinine
3.447 (0.09%) 4.435 (0.16%) 7.806 (0.24%) 8.896 (0.08%)
2,505,014 (0.05%) 6,773,343 (0.14%) 7,846,639 (0.91%) 1,515,788 (0.91%)
0.9995
3.96 × 10−6
8.866
26.866
–
0.9999
7.35 × 10−7
0.009
0.026
–
0.9988
6.18 × 10
−7
0.050
0.15
2.753
1.65 × 10
−5
0.034
0.104
1.211
Neopterin Biopterin Pterin
0.9995
RT — retention time, R2 — coefficient of determination, LOD — limit of detection, LOQ — limit of quantitation, RSD — relative standard deviation.
4. Discussion Pteridines are very important cofactors in the processes of cell metabolism which are excreted in urine. Reduced forms of pteridines are almost non-fluorescent and they undergo oxidation easily. Only fully oxidized forms of unconjugated pteridine species show intensive fluorescence [2,3]. With the aim of increasing the fluorescence signal of reduced pteridines, we had oxidized all the urine samples as a first step and after that we quantified the total amounts of urine pteridines. The intensive fluorescent signal in all urine samples was provided by neopterin and biopterin, in accordance with papers of Rokos [21] and Wachter [22], and at the same time, we detected a weaker but still well identifiable signal for pterin. In our work we assessed pteridine levels in urine from patients with malignant and benign ovarian tumors and compared them with each other and with the levels found in healthy persons (controls). We have recorded the total neopterin levels from oxidized urine samples, which comprise the reduced form (dihydroneopterin) and the naturally occurring oxidized neopterin. We have found that the total neopterin levels were significantly higher in urine samples from patients with malignant (4.3 times) and benign ovarian tumors (2.9 times) relative to the control group. The total neopterin level in patients with a malignant tumor was also significantly higher (1.5 times) than the level of patients with a benign tumor. The observation of increased urinary neopterin in patients with ovarian cancer in the present study is in agreement with previous reports [17,20,23–26], but concentrations of neopterin in the urine of patients with benign ovarian tumors have not yet been presented. Although the examination of benign tumors is still relatively infrequent, all papers published so far as well as our research show higher neopterin levels in patients with malignant tumors
than in patients with benign tumors [24,25,27,28]. It is known that neopterin production in human monocytes/macrophages is elevated after interferon γ stimulation in cases of activated cellular immunity [8,29,30]. Increased neopterin biosynthesis can be also caused by other cytokines, such as tumor necrosis factor α, and alloantigens [4, 31]. Upon stimulation with interferons also monocyte derived dendritic cells were found to produce neopterin in similar concentrations as macrophages [9]. Other human cells may produce neopterin upon stimulation with interferon γ, but to a smaller extent than macrophages [32]. In addition, weak neopterin production by epithelial cells is accompanied by intensive production of tetrahydrobiopterin [9]. Our data showed a sizable increase of neopterin levels in ovarian cancer samples, and a weaker increase in benign ovarian tumor samples compared to controls. As neopterin is produced by several cell types along the specific metabolic pathway and is stimulated variously, its different levels in urine samples of malignant and benign tumors are probably caused by the different immune response and/or the different processes of neopterin production in patients with malign and benign tumors. The total biopterin concentrations obtained in our study correspond to the sum of contributions by tetrahydrobiopterin, dihydrobiopterin, and biopterin (the naturally occurring oxidized biopterin form) in the urine samples [6]. We found that the levels of total biopterin in the urine of patients with malignant or benign ovarian tumors were twice as high as the levels of total biopterin in the urine of healthy women. We did not find any difference between urine samples from patients with benign and malignant ovarian tumor. There is a lack of literary sources dealing with biopterin levels in urine from patients with a benign ovarian tumor as well as with biopterin levels in urine from patients with ovarian cancer. Only few researchers had been estimating biopterin levels in urine of cancer patients, but they used the urine of
Fig. 3. Chromatogram of a urine sample. Absorption detection (250 nm).
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Fig. 4. Chromatogram of a urine sample. Fluorescence detection (excitation 350 nm, emission 450 nm).
patients with different malignancies. Gamagedara et al. [13] measured pteridine levels in healthy persons and patients with a mix of various cancer types. They had observed significantly increased biopterin levels in cancer patients. Earlier publications had presented unchanged biopterin levels [14] or nonsignificantly increased levels of biopterin [12] in patients with a mix of cancer types in comparison with healthy persons. Nowadays it is well known that tetrahydrobiopterin is synthesized to a variable extent in many human tissues, like fibroblasts or endothelial cells [33,34]. However, until now, quantitative changes of biopterin levels were not adequately examined. Cytokines such as interferon γ or tumor necrosis factor-α strongly stimulate the activity of guanosine triphosphate cyclohydrolase I in human cells, yielding a potentiation of intracellular tetrahydrobiopterin concentrations. A functional role for the stimulation of tetrahydrobiopterin biosynthesis by cytokines is the formation of a limiting cofactor required for enzymatic conversion of L-arginine to citrulline and nitric oxide [34]. The synthesis of tetrahydrobiopterin is induced as well as inducible nitric oxide synthase by lipopolysaccharide and/or cytokines [35]. Tetrahydrobiopterin and NADPH are known to modulate nitric oxide synthesis by endothelial cells and thus have the potential to regulate of endothelial cell proliferation [36]. According to our results, the increase of biopterin levels was the same in patients with malignant and benign ovarian tumors, so the presence of an ovarian tumor (malignant or benign) presumably stimulates the same metabolic pathway of tetrahydrobiopterin synthesis. The levels of urine pterin in our study followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels. The correlation between neopterin and pterin levels in urine indicates that pterin is an intermediate product in the degradation metabolic pathway of neopterin.
There are no findings in the literature about pterin concentrations in the urine of patients with benign or malignant ovarian tumors. We can only compare our results with findings of a few studies [13,14,32] that reported an increased level of pterin in human urine from patients with a mix of various cancer types. Pterin does not seem to be as useful in clinical conditions as neopterin, but the assessment of its levels can lead to better quantitative understanding of metabolic pathways of pteridines during various neoplastic diseases.
5. Conclusions We have assessed the levels of neopterin, biopterin, and pterin in urine of patients with malignant and benign ovarian tumors and healthy persons with the purpose of finding the differences in the production of the above mentioned pteridines caused by the presence of benign or malignant tumors. Our data showed a sizable increase in neopterin levels in the urine from ovarian cancer patients, and a weaker increase for patients with benign ovarian tumors compared to healthy persons. This can be caused by a different immune response and/or different processes of neopterin production in patients with malignant and benign tumors.
Table 2 Median values with interquartile range of three pteridine levels excreted in urine by control (healthy subjects) and patients with benign and malignant ovarian tumors. Median
Neopterin
Biopterin
Pterin
Sample
μmol/mmol creatinine
μmol/mmol creatinine
μmol/mmol creatinine
Control
0.056 (0.048–0.080) 0.150** (0.161–0.217) 0.226** (0.163–0.330)
0.096 (0.072–0.132) 0.268** (0.199–0.343) 0.239** (0.187–0.321)
0.020 (0.008–0.040) 0.041* (0.022–0.064) 0.056** (0.034–0.111)
Benign Malignant
**0.001, *0.01 — significance level of the Mann–Whitney U test as used to compare concentration of urine pteridines from patients and healthy subjects.
Fig. 5. Box plot of neopterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001 significance level of the Mann–Whitney U test used to compare medians of neopterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
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References
Fig. 6. Box plot of biopterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001 significance level of the Mann–Whitney U test used to compare medians of biopterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
The increase of biopterin levels was the same for patients with malignant and benign ovarian tumors in comparison with healthy individuals. Thus, the presence of both ovarian tumors (malignant or benign) presumably stimulates the same metabolic pathway of biopterin synthesis. The levels of urine pterin in our study followed a pattern similar to neopterin levels, but their concentrations were about three times lower than the levels of neopterin, which indicates that pterin is an intermediate product in the degradation metabolic pathway of neopterin.
Acknowledgments This study was supported by APVV grant no. 0134-12. This publication is also the result of implementation of the following projects: BIOMAKRO2, ITMS: 26240120027 supported by the Research & Development Operational Program funded by the ERDF.
Fig. 7. Box plot of pterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001, *0.01, †0.05 significance level of the Mann–Whitney U test used to compare medians of pterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jpb
Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals M. Zvarik a, D. Martinicky b, L. Hunakova c, L. Sikurova a,⁎ a b c
Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Computer Science, Comenius University, Bratislava, Slovakia Department of Gynecological Oncology, National Cancer Institute, Bratislava, Slovakia Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 1 April 2015 Received in revised form 17 August 2015 Accepted 16 September 2015 Available online 24 September 2015 Keywords: Fluorescence Pteridine Urine Ovarian tumors
a b s t r a c t Pteridines belong to a class of fluorescent metabolites that are excreted by humans in urine and their concentrations can reflect various pathophysiological states. We quantified the differences in urinary pteridine levels in patients with malignant and benign ovarian tumors and in healthy individuals. Urine samples were centrifuged and supernatants were oxidized by MnO2 before analysis. Levels of neopterin, biopterin, and pterin were assessed by fluorescence analysis of human urine after HPLC separation. We have revealed that the median neopterin levels were higher in urine samples from patients with malignant (0.226 μmol/mmol creatinine) and benign ovarian tumors (0.150 μmol/mmol creatinine) than in healthy subjects (0.056 μmol/mmol creatinine). The median neopterin levels of patients with malignant tumors were higher (1.5-times) than in patients with benign tumors. The median biopterin level in urine of patients with benign ovarian tumors (0.268 μmol/mmol creatinine) was found to be very close to the level in patients with malignant ovarian tumors (0.239 μmol/mmol creatinine), and both were higher than in healthy samples (0.096 μmol/mmol creatinine). The levels of urine pterin followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels. © 2015 Published by Elsevier B.V.
1. Introduction Pteridines are low molecular weight substances belonging to a class of fluorescent metabolites that are excreted by humans in urine. The naturally occurring pteridine derivatives exist in three different oxidation states: tetrahydro-, dihydro-, and fully-oxidized [1]. Fully oxidized pteridines are fluorescent compounds, while the fluorescence of reduced derivatives is much weaker [2,3]. Scientific data available to-date show that in humans, pteridines are synthesized from guanosine triphosphate (GTP) by GTPcyclohydrolase I [4]. GTP cyclohydrolase I converts GTP to 7,8dihydroneopterin-3′-triphosphate. This intermediate is further metabolized by 6-pyruvoyl tetrahydropterin synthase to 6-pyruvoyl tetrahydropterin, which is finally converted by two NADPHdependent reductions to tetrahydrobiopterin, the active hydroxylating compound. These NADPH-dependent hydroxylations are carried out by sepiapterin reductase [5].
⁎ Corresponding author at: Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Computer Science, Mlynska dolina F1, 84248 Bratislava, Slovakia. E-mail addresses: [email protected] (M. Zvarik), [email protected] (D. Martinicky), [email protected] (L. Hunakova), [email protected] (L. Sikurova).
http://dx.doi.org/10.1016/j.jphotobiol.2015.09.019 1011-1344/© 2015 Published by Elsevier B.V.
5,6,7,8-Tetrahydrobiopterin has been recognized as the most important unconjugated pteridine existing in biological human fluids [1]. Biopterin and dihydrobiopterin are the oxidative products of tetrahydrobiopterin [6]. In the human organism, tetrahydrobiopterin is involved in the hydroxylation reactions of phenylalanine, tyrosine and tryptophan. It is also an essential cofactor for the biosynthesis of the neurotransmitters dopamine, noradrenaline and serotonin, and of the reactive free radical nitric oxide. In addition, tetrahydrobiopterin also enters the metabolism of lipids as a cofactor of alkylglycerol monooxygenase (also called glyceryl ether monooxygenase, GEMO). GEMO is the only enzyme known to cleave the ether bond in alkylglycerol ether [7]. Tetrahydrobiopterin provides electrons being oxidized to dihydrobiopterin, which is recycled to tetrahydrobiopterin by action of the dihydropteridine reductase. Nearly all human cells like fibroblasts or endothelial cells produce tetrahydrobiopterin from GTP and only scarce amounts of neopterin species are formed. The only exception being monocytes/macrophages and monocyte derived dendritic cells, in which, due to the low activity of 6-pyruvoyl-tetrahydropterinsynthetase, relevant amounts of neopterin and dihydroneopterin are formed [5]. Several unconjugated pteridines, particularly neopterin, biopterin, xanthopterin, isoxanthopterin and pterin are responsible for blue autofluorescence of human urine [6]. Urinary excretion of these compounds has been found to increase as a result of several disorders such as viral
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infections, intracellular bacteria and parasites, chronic inflammatory disorders, or autoimmune diseases [8,9]. Using various fluorescence techniques, a crucial fact for diagnostics was established: the native blue fluorescence of urine pteridines in oncological patients is different from that in healthy individuals [10–12]. However, those data often came from a mixture of different tissues and tumor types [1,13,14]. Although Han et al. [14] stated that each type of tumor shows its own pattern in changes of pteridine concentrations in urine because different pteridine derivatives may play various roles in different tumor-related diseases, studies where the levels of pteridine derivatives in urine from patients with exclusively one type of tumor are assessed are scarce [15]. Ovarian cancer is the third most common gynecological cancer [16], but there is still lack of studies dealing with the quantification of pteridine levels in patients with ovarian malignancies [17–20], and urine pteridine studies focused on distinguishing between malignant and benign ovarian tumors are completely missing. The quantitative evaluation of pteridines is very important not only for a potential early diagnostics of the disease, but it also helps to explain the differences in metabolism of pteridines in various types of tumors and to tell apart malignant and benign tumors. In this study, we used high performance liquid chromatography (HPLC) analysis with fluorescence detection for quantitative evaluation of three pteridines (neopterin, biopterin, pterin) in urine of patients with ovarian tumors to establish differences between patients with malignant and benign ovarian tumors and healthy individuals. 2. Materials and Methods 2.1. Chemicals 6-Biopterin, D-erythro-neopterin, pterin, creatinine, methanol and water (HPLC grade) were purchased from Sigma-Aldrich Co. Sodium hydroxide, hydrochloric acid, monopotassium phosphate monobasic and manganese dioxide were obtained from Slavus Ltd. (Bratislava, Slovakia).
2.2. Buffer Preparation A 10 mmol/l aqueous solution of KH2PO4 sample buffer was prepared by diluting 10 ml of 1 mol/l KH2PO4 with water and by adding 30 ml of methanol. The buffer was filled with water to a final volume of 1 l and adjusted to pH 4.5 with HCl.
2.4. Urine Sample Preparation This study has been cleared by The National Cancer Institute (Slovakia) Ethics Review Board for human study and patients have signed an informed consent. 32 morning urine samples from fasting normal volunteers and 75 patients were used in this study. All of them were analyzed for pH, protein, glucose, bilirubin, nitrate, hemoglobin, ketones, acetone, and urobilinogen. The presence of red blood cells, white blood cells, casts, epithelial cells and crystals was also tested in these samples at the Department of Clinical Biochemistry, National Cancer Institute, Bratislava, Slovakia. Urine samples were taken from patients who did not undergo chemical or radiation therapy, they were taken before the start of anticancer or antibiotic therapy. The control group volunteers did not take any medications including vitamin supplements. The age distribution was 24–77 years for patients and 23–64 years for healthy subjects. The group of oncological patients was represented by 36 patients with confirmed ovarian malignant tumor (ICD 10 code C56) and 39 patients with ovarian benign tumor (ICD 10 code D27). Urine samples were centrifuged at 3000 rpm for 10 min at room temperature (22 ± 1 °C) and undiluted supernatants were used for analysis. All samples were stored in a freezer at −35 °C. Prior to analysis, the samples were removed from the freezer and brought to room temperature. Human urine samples were subjected to HPLC analysis after MnO2 oxidation. We pipetted 1 ml of fresh random urine into a centrifugation vial and adjusted pH to 1.0–1.5 with 40 μl of 6 mol/l HCl. After that we added 20 mg of MnO2 and shook for 5 min at room temperature. Then, we centrifuged for 5 min at 3000 ×g and immediately transferred clear supernatant into a vial. The vial was wrapped in aluminium foil to protect its content from light. 2.5. High Performance Liquid Chromatography System The chromatographic studies were performed on a modular HPLC system Prominence 20A (Shimadzu Co.), equipped with degasser (DGU-20A5), solvent delivery unit with quaternary pump (LC-20AD), autosampler with a sample cooler (SIL-20AC), column oven (CTO20AC), UV–VIS absorption detector (SPD-20A), fluorescence detector (RF-10AXL), system controller (CBM-20A) and the LC solutions software (1.25 SP1) to control the instrument, data acquisition and data analysis. The analytical columns used were Nucleosil® C18, 150 mm × 3.2 mm, 5 μm particle size (Supelco Analytical). 2.6. High Performance Liquid Chromatography Analysis
2.3. Standard Preparation Pteridine stock solutions (0.01% w/v) were prepared by dissolving separately 1 mg of neopterin, biopterin, or pterin in 1 ml 0.1 mol/l NaOH. We kept solutions in an ultrasound bath for 30 s, then we added 9 ml 0.1 mol/l HCl, and kept it in an ultrasound bath for another 30 s. Pteridine working solutions (0.001% w/v) were prepared by diluting 1 ml of each 0.01% pteridine stock solution with 9 ml 0.05 mol/l HCl. Urine standard mixture was prepared by pipetting 324 μl resp. 2530 μl (pterin resp. neopterin and biopterin) of 0.001% pteridine working solutions in a 20-ml flask and filling with 0.05 mol/l HCl to the mark. A dilution series of this mixture were used to generate calibration curves (1, 2, 3, 4 and 5 μmol/l for neopterin and biopterin, respectively; 0.2, 0.4, 0.6, 0.8 and 1 μmol/l for pterin). Standard creatinine stock solution was prepared by dissolving 45 mg creatinine in 10 ml 0.05 mol/l solution HCl to make the creatinine concentration of 40 mmol/l. This standard stock solution was diluted to an appropriate concentration with 0.05 mol/l solution of HCl (for calibration 5, 10, 20, 30 and 40 mmol/l). The standard solutions were stored at −35 °C.
Oxidized samples of urine were eluted isocratically with buffer solution (10 mmol/l aqueous solution of KH2PO4, 3% methanol) at a flow of 0.6 ml/min. The injected volume sample was 5 μl. After 20 min, when pteridines were eluted, the next sample could be injected. After 5 injections of biological samples, the columns were rinsed with aqueous solution of methanol (1:1) followed by buffer solution, to condition them for the next series of samples. The system temperature was adjusted to 25 °C. Detection was performed with photometric detector at 250 nm for creatinine analysis, and fluorimetric detector at 450 nm (excitation at 350 nm) for pteridine analysis, connected in serial.
2.7. Statistical Analysis Pteridine concentrations were determined by linear regression against experimentally generated calibration curves by LC solutions software (Shimadzu Co.). The collected data were graphically and statistically processed by the R software. The Mann–Whitney U test was used to compare medians of pteridine urine levels from healthy persons and patients.
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Fig. 1. Chromatogram of the creatinine standard (10 mmol/l). Absorption detection (250 nm).
3. Results Full oxidization of pteridines in the urine samples was selected for the reliability of pteridine fluorescence quantification. The levels of urine pteridines were normalized to urine creatinine concentrations so that fluctuations in urine concentration were minimized. The linearity of the HPLC method was evaluated by using standard creatinine solutions and standard mixtures of neopterin, biopterin, and pterin with different concentrations. The standard mixtures were injected into the HPLC by triplicate to obtain their elution order (Figs. 1 and 2). The acquired detection limit, linear equations and R2 values are given in Table 1. HPLC examination of human urine samples by UV absorption detection confirmed the narrow and intensive signal for creatinine (Fig. 3). Fluorescence chromatograms of all urine samples showed multiple peaks within a 15 min. time frame, where the peaks of neopterin and biopterin were convincingly identified and pterin provided weaker signal (Fig. 4). The levels of the all three pteridines studied in the urine from patients with malignant ovarian tumors were found to be significantly higher (p b 0.001) than those for the control group (healthy subjects) (Table 2). Moreover, the median value of urine neopterin concentration for cancer patients (0.226 μmol/mmol creatinine) exceeded the upper limit of healthy subjects (Fig. 5). The median levels of neopterin were less but significantly elevated (p b 0.001) also in the urine samples
from patients with benign ovarian tumor (0.150 μmol/mmol creatinine) as compared to controls (0.056 μmol/mmol creatinine). In addition, the level of urine neopterin was significantly elevated (p b 0.001) in patients with malignant ovarian tumors (0.226 μmol/mmol creatinine) as compared to those with benign ovarian tumors (0.150 μmol/mmol creatinine). Urine biopterin levels showed a different pattern in patients with benign ovarian tumor (0.268 μmol/mmol creatinine), acquiring nearly identical value to that found in samples from patients with malignant ovarian tumor (0.239 μmol/mmol creatinine); these values were significantly higher (p b 0.001) compared to healthy samples (0.096 μmol/mmol creatinine) (Fig. 6). Levels of urine pterin showed a pattern similar to neopterin, however pterin concentrations were about 3 times lower than those of neopterin and disease-related changes were observed with lower significance (Fig. 7). The median urine pterin levels for patients with malignant ovarian tumor (0.056 μmol/mmol creatinine) were significantly higher (p b 0.001) than the control level (0.020 μmol/mmol creatinine) and also than the level found in patients with benign ovarian tumors (0.041 μmol/mmol creatinine), but only with p b 0.05. The urine pterin level for patients with benign ovarian tumor was significantly higher (p b 0.01) than in healthy subjects. Spearman's rank correlation coefficients were found to be 0.715 for neopterin/ biopterin, 0.663 for neopterin/pterin, and 0.687 for biopterin/pterin.
Fig. 2. Chromatogram of the standard mixture of three pteridine standards. Fluorescence detection (excitation 350 nm, emission 450 nm).
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Table 1 Validation parameters of HPLC method. Compound
RT/min (RSD)
Area/mV·min (RSD)
R2
Slope
LOD (μmol/l)
LOQ (μmol/l)
Selectivity (α)
Creatinine
3.447 (0.09%) 4.435 (0.16%) 7.806 (0.24%) 8.896 (0.08%)
2,505,014 (0.05%) 6,773,343 (0.14%) 7,846,639 (0.91%) 1,515,788 (0.91%)
0.9995
3.96 × 10−6
8.866
26.866
–
0.9999
7.35 × 10−7
0.009
0.026
–
0.9988
6.18 × 10
−7
0.050
0.15
2.753
1.65 × 10
−5
0.034
0.104
1.211
Neopterin Biopterin Pterin
0.9995
RT — retention time, R2 — coefficient of determination, LOD — limit of detection, LOQ — limit of quantitation, RSD — relative standard deviation.
4. Discussion Pteridines are very important cofactors in the processes of cell metabolism which are excreted in urine. Reduced forms of pteridines are almost non-fluorescent and they undergo oxidation easily. Only fully oxidized forms of unconjugated pteridine species show intensive fluorescence [2,3]. With the aim of increasing the fluorescence signal of reduced pteridines, we had oxidized all the urine samples as a first step and after that we quantified the total amounts of urine pteridines. The intensive fluorescent signal in all urine samples was provided by neopterin and biopterin, in accordance with papers of Rokos [21] and Wachter [22], and at the same time, we detected a weaker but still well identifiable signal for pterin. In our work we assessed pteridine levels in urine from patients with malignant and benign ovarian tumors and compared them with each other and with the levels found in healthy persons (controls). We have recorded the total neopterin levels from oxidized urine samples, which comprise the reduced form (dihydroneopterin) and the naturally occurring oxidized neopterin. We have found that the total neopterin levels were significantly higher in urine samples from patients with malignant (4.3 times) and benign ovarian tumors (2.9 times) relative to the control group. The total neopterin level in patients with a malignant tumor was also significantly higher (1.5 times) than the level of patients with a benign tumor. The observation of increased urinary neopterin in patients with ovarian cancer in the present study is in agreement with previous reports [17,20,23–26], but concentrations of neopterin in the urine of patients with benign ovarian tumors have not yet been presented. Although the examination of benign tumors is still relatively infrequent, all papers published so far as well as our research show higher neopterin levels in patients with malignant tumors
than in patients with benign tumors [24,25,27,28]. It is known that neopterin production in human monocytes/macrophages is elevated after interferon γ stimulation in cases of activated cellular immunity [8,29,30]. Increased neopterin biosynthesis can be also caused by other cytokines, such as tumor necrosis factor α, and alloantigens [4, 31]. Upon stimulation with interferons also monocyte derived dendritic cells were found to produce neopterin in similar concentrations as macrophages [9]. Other human cells may produce neopterin upon stimulation with interferon γ, but to a smaller extent than macrophages [32]. In addition, weak neopterin production by epithelial cells is accompanied by intensive production of tetrahydrobiopterin [9]. Our data showed a sizable increase of neopterin levels in ovarian cancer samples, and a weaker increase in benign ovarian tumor samples compared to controls. As neopterin is produced by several cell types along the specific metabolic pathway and is stimulated variously, its different levels in urine samples of malignant and benign tumors are probably caused by the different immune response and/or the different processes of neopterin production in patients with malign and benign tumors. The total biopterin concentrations obtained in our study correspond to the sum of contributions by tetrahydrobiopterin, dihydrobiopterin, and biopterin (the naturally occurring oxidized biopterin form) in the urine samples [6]. We found that the levels of total biopterin in the urine of patients with malignant or benign ovarian tumors were twice as high as the levels of total biopterin in the urine of healthy women. We did not find any difference between urine samples from patients with benign and malignant ovarian tumor. There is a lack of literary sources dealing with biopterin levels in urine from patients with a benign ovarian tumor as well as with biopterin levels in urine from patients with ovarian cancer. Only few researchers had been estimating biopterin levels in urine of cancer patients, but they used the urine of
Fig. 3. Chromatogram of a urine sample. Absorption detection (250 nm).
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Fig. 4. Chromatogram of a urine sample. Fluorescence detection (excitation 350 nm, emission 450 nm).
patients with different malignancies. Gamagedara et al. [13] measured pteridine levels in healthy persons and patients with a mix of various cancer types. They had observed significantly increased biopterin levels in cancer patients. Earlier publications had presented unchanged biopterin levels [14] or nonsignificantly increased levels of biopterin [12] in patients with a mix of cancer types in comparison with healthy persons. Nowadays it is well known that tetrahydrobiopterin is synthesized to a variable extent in many human tissues, like fibroblasts or endothelial cells [33,34]. However, until now, quantitative changes of biopterin levels were not adequately examined. Cytokines such as interferon γ or tumor necrosis factor-α strongly stimulate the activity of guanosine triphosphate cyclohydrolase I in human cells, yielding a potentiation of intracellular tetrahydrobiopterin concentrations. A functional role for the stimulation of tetrahydrobiopterin biosynthesis by cytokines is the formation of a limiting cofactor required for enzymatic conversion of L-arginine to citrulline and nitric oxide [34]. The synthesis of tetrahydrobiopterin is induced as well as inducible nitric oxide synthase by lipopolysaccharide and/or cytokines [35]. Tetrahydrobiopterin and NADPH are known to modulate nitric oxide synthesis by endothelial cells and thus have the potential to regulate of endothelial cell proliferation [36]. According to our results, the increase of biopterin levels was the same in patients with malignant and benign ovarian tumors, so the presence of an ovarian tumor (malignant or benign) presumably stimulates the same metabolic pathway of tetrahydrobiopterin synthesis. The levels of urine pterin in our study followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels. The correlation between neopterin and pterin levels in urine indicates that pterin is an intermediate product in the degradation metabolic pathway of neopterin.
There are no findings in the literature about pterin concentrations in the urine of patients with benign or malignant ovarian tumors. We can only compare our results with findings of a few studies [13,14,32] that reported an increased level of pterin in human urine from patients with a mix of various cancer types. Pterin does not seem to be as useful in clinical conditions as neopterin, but the assessment of its levels can lead to better quantitative understanding of metabolic pathways of pteridines during various neoplastic diseases.
5. Conclusions We have assessed the levels of neopterin, biopterin, and pterin in urine of patients with malignant and benign ovarian tumors and healthy persons with the purpose of finding the differences in the production of the above mentioned pteridines caused by the presence of benign or malignant tumors. Our data showed a sizable increase in neopterin levels in the urine from ovarian cancer patients, and a weaker increase for patients with benign ovarian tumors compared to healthy persons. This can be caused by a different immune response and/or different processes of neopterin production in patients with malignant and benign tumors.
Table 2 Median values with interquartile range of three pteridine levels excreted in urine by control (healthy subjects) and patients with benign and malignant ovarian tumors. Median
Neopterin
Biopterin
Pterin
Sample
μmol/mmol creatinine
μmol/mmol creatinine
μmol/mmol creatinine
Control
0.056 (0.048–0.080) 0.150** (0.161–0.217) 0.226** (0.163–0.330)
0.096 (0.072–0.132) 0.268** (0.199–0.343) 0.239** (0.187–0.321)
0.020 (0.008–0.040) 0.041* (0.022–0.064) 0.056** (0.034–0.111)
Benign Malignant
**0.001, *0.01 — significance level of the Mann–Whitney U test as used to compare concentration of urine pteridines from patients and healthy subjects.
Fig. 5. Box plot of neopterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001 significance level of the Mann–Whitney U test used to compare medians of neopterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
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References
Fig. 6. Box plot of biopterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001 significance level of the Mann–Whitney U test used to compare medians of biopterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
The increase of biopterin levels was the same for patients with malignant and benign ovarian tumors in comparison with healthy individuals. Thus, the presence of both ovarian tumors (malignant or benign) presumably stimulates the same metabolic pathway of biopterin synthesis. The levels of urine pterin in our study followed a pattern similar to neopterin levels, but their concentrations were about three times lower than the levels of neopterin, which indicates that pterin is an intermediate product in the degradation metabolic pathway of neopterin.
Acknowledgments This study was supported by APVV grant no. 0134-12. This publication is also the result of implementation of the following projects: BIOMAKRO2, ITMS: 26240120027 supported by the Research & Development Operational Program funded by the ERDF.
Fig. 7. Box plot of pterin levels in benign (n = 39) and malignant (n = 36) ovarian tumor urine samples, and control urine samples (n = 32). **0.001, *0.01, †0.05 significance level of the Mann–Whitney U test used to compare medians of pterin urine levels from control (healthy subjects) and patients with benign or malignant tumors.
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