Identification of hypoxanthine as a urine marker for non-Hodgkin lymphoma by low-mass-ion profiling
© Yoo et al; licensee BioMed Central Ltd. 2010
Received: 25 May 2009
Accepted: 23 February 2010
Published: 23 February 2010
Non-Hodgkin lymphoma (NHL) is a hematologic malignancy for which good diagnostic markers are lacking. Despite continued improvement in our understanding of NHL, efforts to identify diagnostic markers have yielded dismal results. Here, we translated low-mass-ion information in urine samples from patients with NHL into a diagnostic marker.
To minimize experimental error, we tested variable parameters before MALDI-TOF analysis of low-mass ions in urine. Urine from 30 controls and 30 NHL patients was analyzed as a training set for NHL prediction. All individual peak areas were normalized to total area up to 1000 m/z. The training set analysis was repeated four times. Low-mass peaks that were not affected by changes in experimental conditions were collected using MarkerView™ software. Human Metabolome Database (HMDB) searches and ESI LC-MS/MS analyses were used to identify low-mass ions that exhibited differential patterns in control and NHL urines. Identified low-mass ions were validated in a blinded fashion in 95 controls and 66 NHL urines to determine their ability to discriminate NHL patients from controls.
The 30 highest-ranking low-mass-ion peaks were selected from the 60-urine training set, and three low-mass-ion peaks with high intensity were selected for identification. Of these, a 137.08-m/z ion showed lower mass-peak intensity in urines of NHL patients, a result that was validated in a 161-urine blind validation set (95 controls and 66 NHL urines). The 130.08-m/z ion was identified from HMDB searches and ESI LC-MS/MS analyses as hypoxanthine (HX). The HX concentration in urines of NHL patients was significantly decreased (P < 0.001) and was correlated with the mass-peak area of the 137.08-m/z ion. At an HX concentration cutoff of 17.4 μM, sensitivity and specificity were 79.2% and 78.4%, respectively.
The present study represents a good example of low-mass-ion profiling in the setting of disease screening using urine. This technique can be a powerful non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, HX identified in the study may be a useful single urine marker for NHL screening.
Non-Hodgkin lymphomas (NHL) are a heterogeneous group of malignancies that arise from lymphoid tissue. They exhibit varied clinical and biological features , and their incidence has been increasing over the past several decades . The past decade has seen enormous changes in our understanding of lymphomas, including the identification of better prognostic factors . However, results from efforts to identify good diagnostic factors have been disappointing. Lactate dehydrogenase has been used as an NHL marker , but its accuracy in diagnosis has been unsatisfactory. Thus, finding specific tumor markers that are useful for diagnosing and monitoring NHL remains a high priority. In the present study, we introduce a new NHL diagnostic marker obtained by translating the information in low-mass ions (i.e., < 1000 m/z) in urine samples from lymphoma patients.
Urine from patients with NHL
The characteristics of 96 NHL patients
No. of Patients (%)
Age, years (median, range)
Histologic type (WHO)
Diffuse large B cell lymphoma
Mantle cell lymphoma
T cell lineage
Bone marrow involvement
MALDI-TOF analytical conditions for collecting low-mass ions in urine
Urine samples were mixed (1:12) with an α-cyano-4-hydroxycinnamic acid solution in 50% acetonitrile/0.1% trifluoroacetic acid (TFA). Differences between normal and cancer urine samples were determined using a 4700 Proteomic Analyzer (Applied Biosystems, Foster City, CA, USA). The mass-spectral data represent the average of 20 accumulated spectra.
Low-mass ion selection and statistical analysis
All MALDI mass spectra, formatted as *.t2d files, were analyzed with MarkerView™ Software version 1.2 (Applied Biosystems/MDS Sciex, Toronto, Canada). The optimized parameters used to compare low-mass peaks in urines from controls and NHL patients were as follow: Mass tolerance, 100 ppm; minimum required response, 100; maximum number of peaks, 5000; normalization, by total area sums. After collecting information from MALDI mass spectra, principal component analyses (PCA) and t-tests were used to select low-mass ions with differential peak intensities in urines from controls and NHL patients.
ESI-MS/MS for low-mass ion analysis
MALDI-TOF was not suitable for comparing MS/MS patterns of low-mass ions, so ESI-MS/MS was employed. The mass spectrometer was set for ESI in positive mode. A syringe pump was used to introduce the calibration solution for automatic tuning and calibration of the LTQ-XL (Thermo Fisher Scientific Inc., Waltham, MA) in ESI positive-ion mode. Standard solutions (1 μM hypoxanthine) were infused directly into the ionization source of the mass spectrometer using a syringe pump (1.0 μL/min) without chromatographic separation. The spray voltage was set at +1.1 kV; the temperature of the capillary was set at 200°C; the capillary voltage was set at +20 V; the tube lens voltage was set at +100 V; and the auxiliary gas was set to zero. Full-scan experiments were performed to linear trap in the range, 100-200 m/z. Systematic MS/MS experiments were performed by changing the relative collisional energy and monitoring the intensities of the fragment ions. MS/MS data were acquired from urine samples.
Dtermination of hypoxanthine and xanthine in urines
The concentration of hypoxanthine and xanthine in urines was determined using the Amplex® Red Xanthine/Xanthine Oxidase Assay Kit (Molecular Probes, Inc., Eugene, OR), according to the manufacturer's instructions.
Experimental conditions for MALDI MS analysis of low-mass ions in urine
Selection of low-mass ions differentially present in control and NHL urines
Identifications for the 137.1 m/z ion in urine as hypoxanthine
Metabolites with 137.07 ± 0.05 m/z in a positive-mode mass detection
Adduct MW (Da) [Matching HMDB MW]
MW Difference (Da) [QueryMass - AdductMass]
Clinicopathological relevance of NHL discriminating low-mass ions in urine
Neither of the two NHL-discriminating low-mass ions, hypoxanthine and xanthine, was significantly correlated with individual histologic types, cancer stage, extra-nodal involvement, Eastern Cooperative Oncology Group score (ECOG performance), international prognostic index (low, low/intermediate vs. high/intermediate, high), bone marrow involvement (absent vs. present), first response to chemotherapy (complete response, partial response vs. stable disease, progressive disease) or survival (data not shown).
Previous efforts to find diagnostic factors for NHL have yielded disappointing results compared with studies reporting prognostic factors with clinicopathological correlations [5, 6]. Our previous study showed that the urine level of IL-8 normalized to creatinine could be a possible biomarker with the capacity to discriminate NHL patients from normal controls; thus, we expected that urine might be a valuable biological source for diagnostic markers for NHL .
In the present study, we sought to develop a new diagnostic approach for NHL, using MALDI-MS analysis to translate the information of low-mass ions (i.e., < ~1000 m/z) present in urine samples into a tool capable of discriminating NHL patients from normal individuals. There are two motivations for our focus on this low-mass range. The first is the existence of valuable information in the low-mass range, analyzed by mass spectrometry, that has not yet been systematically exploited. Second, ions in the low-mass range provide an enormous amount of information about biological changes that originate from alterations in gene and protein expression. We hypothesized that a new non-invasive cancer-screening protocol could be established if low-mass-ion data were properly collected, statistically translated, and analyzed by MALDI-MS.
Interestingly, a low-mass ion appeared at 137.08 m/z that was significantly different in urine samples from NHL patients and controls (Figure 2B &2C). Searching the Human Metabolome Database (HMDB) yielded a list of candidate metabolites corresponding to the 137.08-m/z ion (Table 2). An ESI-MS/MS analysis of low-mass ions in urine identified 137.08 m/z as hypoxanthine (Figure 3B and 3C). Consistent with this identification, the concentration of hypoxanthine in urine compared favorably with the MALDI-MS profile of the 137.08-m/z ion (Figure 3C).
The Low-mass ion profiling is absolutely depends on the accurate mass measuring technology. Recent mass spectrometers employing either MALDI-, or ESI-based technology provide very accurate mass information. Using this advanced technology, we herein were able to suggest a possible application of low-mass ion profiling for cancer screening. However, a couple of problems are still remained. First, software for low-mass ion profiling is still incomplete. During the normalization process of mass spectra obtained from each individual samples, software sometimes collects the noise on the mass spectrum as a low-intensity peaks with discriminating power. To prevent this nose picking, low-mass ions ranked by software have to be checked again on the raw mass spectra. Furthermore, the low-mass ions with low-intensity, even they have a great discriminating power, may not be proper for identification. This is the reason why the low-mass ion with 137.08 m/z was selected for identification since it showed highest intensity among the low-mass ions ranked within 30th (Figure 2B).
In patients with gastric cancer or colorectal cancer, purine bases in plasma have been reported to increase in association with a decrease in purine base excretion . Whether hypoxanthine and xanthine levels are altered in urine samples from gastrointestinal cancer patients is still a matter of debate . In plasma from children with acute lymphoblastic leukemia or NHL, hypoxanthine levels were reported to be higher than those in healthy adult controls; these elevated plasma hypoxanthine levels decreased after methotrexate infusion . However, a change in urine levels of hypoxanthine has not yet been reported.
At present, we are unable to explain the underlying mechanism for the change in hypoxanthine, but one possibility may be found in alterations of purine metabolism that occur during tumor development. Intracellular concentrations of hypoxanthine and xanthine are inversely related to adenylate energy changes and, therefore, to the energy currency of cellular ATP . Recently, a classical antifolate has been shown to possess cytotoxic activity against human prostatic cancer cell lines that lacked hypoxanthine, whereas growth was maintained in tumors with hypoxanthine . We reasoned that the level of hypoxanthine in NHL urines might decrease due to consumption by tumor cells. However, a recent study has shown that urinary hypoxanthine is significantly increased when tumor development in mesothelioma-transplanted nude mice was maximized . In addition, changes in the activity or expression of enzymes involved in hypoxanthine or xanthine metabolism, which might affect hypoxanthine and xanthine levels in NHL urines, cannot be ruled out. For example, xanthine oxidoreductase, a key enzyme in the degradation of DNA and RNA, is associated with histological grade of differentiation and extent of disease in colorectal cancer , as well as the migratory activity of human breast cancer cells [14, 15].
The present study represents a good example of low-mass-ion profiling in the setting of disease-screening using urine samples. This technique can be a powerful, non-invasive diagnostic tool with high sensitivity and specificity for NHL screening. Furthermore, hypoxanthine identified in this study may be a useful single urine marker for NHL screening. However, the biochemical mechanism responsible for the decreased levels of hypoxanthine and xanthine in urine samples from NHL patients remains to be elucidated.
This work was financially supported by research grants from the National Cancer Center, Republic of Korea (HSE; 0810440 & BCY; 1010050), the Innovative Research Institute for Cell Therapy, Republic of Korea (HSE; A062260), and the Ministry of Knowledge, Economy & Industrial Technology Development, Republic of Korea (BCY; 10032113).
- Alexander DD, Mink PJ, Adami HO, Chang ET, Cole P, Mandel JS, Trichopoulos D: The non-Hodgkin lymphomas: a review of the epidemiologic literature. Int J Cancer. 2007, 120 (Suppl 12): 1-39. 10.1002/ijc.22719.View ArticlePubMedGoogle Scholar
- Morrison VA: Non-Hodgkin's lymphoma in the elderly. Part 1: Overview and treatment of follicular lymphoma. Oncology (Williston Park). 2007, 21: 1104-1110.Google Scholar
- Coiffier B: Treatment of non-Hodgkin's lymphoma: a look over the past decade. Clin Lymphoma Myeloma. 2006, 7 (Suppl 1): S7-13. 10.3816/CLM.2006.s.002.View ArticlePubMedGoogle Scholar
- Nicolaides C, Fountzilas G, Zoumbos N, Skarlos D, Kosmidis P, Pectasides D, Karabelis A, Giannakakis T, Symeonidis A, Papadopoulos A, Antoniou F, Pavlidis N: Diffuse large cell lymphomas: identification of prognostic factors and validation of the International Non-Hodgkin's Lymphoma Prognostic Index. A Hellenic Cooperative Oncology Group Study. Oncology. 1998, 55: 405-415. 10.1159/000011886.View ArticlePubMedGoogle Scholar
- Ozgüroglu M, Turna H, Demir G, Döventas A, Demirelli F, Mandel NM, Büyükünal E, Serdengeçti S, Berkarda B: Usefulness of the epithelial tumor marker CA-125 in non-Hodgkin's lymphoma. Am J Clin Oncol. 1999, 22: 615-618. 10.1097/00000421-199912000-00014.View ArticlePubMedGoogle Scholar
- Mihaljević B, Nedeljkov-Janćić R, Cemerikić-Martinović V, Babić D, Colović M: Ki-67 proliferative marker in lymph node aspirates of patients with non-Hodgkin's lymphoma. Med Oncol. 2006, 23: 83-89. 10.1385/MO:23:1:83.View ArticlePubMedGoogle Scholar
- Lee HL, Eom HS, Yun T, Kim HJ, Park WS, Nam BH, Moon-Woo S, Lee DH, Kong SY: Serum and urine levels of interleukin-8 in patients with non-Hodgkin's lymphoma. Cytokine. 2008, 43: 71-75. 10.1016/j.cyto.2008.04.004.View ArticlePubMedGoogle Scholar
- Vannoni D, Porcelli B, Caldarone R, Garzi A: Purine metabolism in human tumors. Medicina (Firenze). 1989, 9: 51-54.Google Scholar
- Hashimoto H, Kubota M, Shimizu T, Kasai Y, Sano H, Adachi S, Akiyama Y, Mikawa H: Effect of high-dose methotrexate on plasma hypoxanthine and uridine levels in patients with acute leukemia or non-Hodgkin lymphoma in childhood. Leukemia. 1992, 6: 1199-1202.PubMedGoogle Scholar
- Harkness RA: Hypoxanthine, xanthine and uridine in body fluids, indicators of ATP depletion. J Chromatogr. 1988, 429: 255-278. 10.1016/S0378-4347(00)83873-6.View ArticlePubMedGoogle Scholar
- Obajimi O, Melera PW: The depletion of cellular ATP by AG2034 mediates cell death or cytostasis in a hypoxanthine-dependent manner in human prostate cancer cells. Cancer Chemother Pharmacol. 2008, 62: 215-226. 10.1007/s00280-007-0593-6.View ArticlePubMedGoogle Scholar
- Buhl L, Dragsholt C, Svendsen P, Hage E, Buhl MR: Urinary hypoxanthine and pseudouridine as indicators of tumor development in mesothelioma-transplanted nude mice. Cancer Res. 1985, 45 (3): 1159-1162.PubMedGoogle Scholar
- Linder N, Martelin E, Lundin M, Louhimo J, Nordling S, Haglund C, Lundin J: Xanthine oxidoreductase - clinical significance in colorectal cancer and in vitro expression of the protein in human colon cancer cells. Eur J Cancer. 2009, 45: 648-655. 10.1016/j.ejca.2008.10.036.View ArticlePubMedGoogle Scholar
- Fini MA, Orchard-Webb D, Kosmider B, Amon JD, Kelland R, Shibao G, Wright RM: Migratory activity of human breast cancer cells is modulated by differential expression of xanthine oxidoreductase. J Cell Biochem. 2008, 105: 1008-1026. 10.1002/jcb.21901.View ArticlePubMedPubMed CentralGoogle Scholar
- Linder N, Lundin J, Isola J, Lundin M, Raivio KO, Joensuu H: Down-regulated xanthine oxidoreductase is a feature of aggressive breast cancer. Clin Cancer Res. 2005, 11: 4372-4381. 10.1158/1078-0432.CCR-04-2280.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/55/prepub
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