Abstract
Pteridines and their derivatives function as intermediates in the metabolism of several vitamins and cofactors, and their relevance to disease has inspired new efforts to study their roles as disease biomarkers. Recent analytical advances, such as the emergence of sensitive mass spectrometry techniques, new workflows for measuring pteridine derivatives in their native oxidation states and increased multiplexing capacities for the simultaneous determination of many pteridine derivatives, have enabled researchers to explore the roles of urinary pteridines as disease biomarkers at much lower levels with greater accuracy than with previous technologies or methods. As a result, urinary pteridines are being increasingly studied as putative cancer biomarkers with promising results being reported from exploratory studies. In addition, the role of urinary neopterin as a universal biomarker for immune system activation is being investigated in new diseases where it is anticipated to become a useful supplementary marker in clinical diagnostic settings. In summary, this review provides an overview of recent developments in the clinical study of urinary pteridines as disease biomarkers, covers the most promising aspects of advanced analytical techniques being developed for the determination of urinary pteridines and discusses the major challenges associated with implementing pteridine biomarkers in clinical laboratory settings.
Introduction
Unconjugated pteridines and their derivatives function as intermediates in the metabolism of several vitamins and cofactors, and their biological concentrations have been shown to become altered with varied pathophysiological processes. Their relevance to disease has made pteridines a promising panel of molecular biomarkers for facilitating disease detection and diagnosis, predicting disease outcome, guiding treatment selection and monitoring therapeutic response. Although altered pteridine levels have been reported in different biological fluids, including serum [1], cerebrospinal fluid [2] and urine [3], their clinical study as putative disease biomarkers, both historical and recent, has focused primarily on their urinary concentrations for several reasons [4]. First, there has been a strong historical precedent to investigate urinary levels of pteridines owing to their original isolation in urine and the resulting analytical methods that were developed for their quantitation in urine. Second, urine presents clinicians and researchers with several key advantages when compared with other specimen types. Namely, tissue and serum specimens must be sampled using invasive procedures and their complex sample matrices can present analytical challenges requiring complicated sample preparation and separation techniques. Alternative specimen types that can be sampled noninvasively, such as exhaled breath, feces, saliva and urine, have received increased attention in recent years, with urine having the highest applicability for pteridine biomarkers given that pteridines are excreted primarily in urine [5], [6]. Finally, the advent of new analytical technologies alongside an improved understanding on disease pathophysiology have opened new areas for the exploration of urinary pteridines as disease biomarkers [4]. For instance, the emergence of metabolomics approaches for biomarker discovery and their inherent applicability to urinary metabolites have provided researchers with new preparative, analytical and statistical workflows for studying urinary pteridines [7]. These new tools have greatly facilitated inquiries into the biological and clinical aspects of pteridines in humans in recent years.
From a historical perspective, research on urinary pteridines in particular has played a key role in the elucidation of their biological functions and clinical significance. As early as 1949, Norris and Majnarich were examining the effects of urinary xanthopterin and so-called anti-xanthopterins on neoplastic cell proliferation [8] with their study findings being quickly met with controversy [9]. The next 20 years would see the successful isolation of xanthopterin [10], biopterin [11], isoxanthopterin [12] and neopterin [13] from human urine. By the 1970s, the first quantitative methods for urinary pteridines using liquid and gas chromatography, mass spectrometry and fluorescence techniques were reported [14], [15], [16]. Immediately following the development of these early analytical methods came the first clinical studies to describe altered levels of pteridines in disease and especially cancer [15], [17], [18], [19], [20]. However, the search for pteridine cancer biomarkers was initially frustrated by limited analytical capabilities and understanding of the biological role of pteridines in cancer development and progression available at that time. Meanwhile, advances in elucidating the biological roles of biopterin and neopterin derivatives shifted focus onto their clinical applications as putative disease biomarkers and the development of specialized techniques for their detection [21], [22], [23]. More recently, the introduction of new robust analytical techniques suitable for comprehensive profiling of urinary pteridines has enabled new investigations in the biological function and clinical applicability of many pteridine derivatives in human health and disease.
In this review, we provide an overview of recent developments in the clinical study of urinary pteridines as disease biomarkers, cover the most promising aspects of advanced analytical techniques being developed for the determination of urinary pteridines and discuss the major challenges associated with implementing pteridine biomarkers in clinical laboratory settings. This paper updates an earlier, and highly recommended, review made by Kośliński et al. [24] by focusing on significant research contributions made in the past 5 years to the study of urinary pteridines as disease biomarkers.
Pteridine metabolism and disease
Limited understanding on the metabolic pathways involved with pteridine biosynthesis and degradation in humans continues to complicate the study of urinary pteridines as putative disease biomarkers. Although pteridine metabolism has been studied elaborately in reptiles [25], amphibians [26], birds [27] and in other lower organisms [28], [29], given its role in pigmentation and de novo synthesis of folate, its elucidation in mammals and especially humans, remains incomplete. The extent of pteridine metabolism in humans that is currently understood is limited to the biosynthetic pathways of 5,6,7,8-tetrahydrobiopterin (BH4), neopterin and their closely related derivatives. Briefly, BH4 is an essential cofactor for several notable enzymes including three aromatic amino acid hydroxylases which catalyze the degradation of phenylalanine and the biosynthesis of several neurotransmitters [23], nitric oxide synthase which catalyzes the formation of gaseous nitric oxide involved with vasodilation and neuroprotection [30], and alkylglycerol monooxygenase which mediates biosynthesis of ether lipids that function as structural components in membranes, chemical signaling agents and as antioxidants [31], [32]. BH4 is synthesized de novo by the conversion of guanosine triphosphate (GTP) to 7,8-dihydroneopterin phosphate via GTP cyclohydrolase I, followed by the successive conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvol-5,6,7,8-tetrahydrobiopterin via 6-pyruvol-5,6,7,8-tetrahydropterin synthase and its subsequent conversion to BH4 by sepiapterin reductase [23]. The catalytic activities of aromatic amino acid hydroxylases and alkylglycerol monooxygenase result in the oxidation of BH4 to BH4-4a-carbinolamine. BH4 can be regenerated from BH4-4a-carbinolamine by either subsequent dehydration and reduction via pterin-4a-carbinolamine dehydratase and dihydropteridine reductase, respectively, or spontaneous dehydration to 6,7-dihydrobiopterin and its subsequent reduction to BH4 by 6,7-dihydropteridine reductase [23].
BH4 deficiency can be caused by various mutations in the genes for the enzymes involved in its biosynthesis and regeneration. BH4 deficiencies can present clinically as a heterogeneous group of diseases and can be detected on the basis of the specific cause. For example, loss of phenylalanine hydroxylase activity caused by low levels of BH4 can result in hyperphenylalaninemia, which is associated with progressive intellectual impairment accompanied by a myriad of additional symptoms [33], [34]. Substantial evidence also suggests that limited vascular BH4 bioavailability causes uncoupling of nitric oxide production from arginine oxidation, resulting in the generation of superoxide radicals and contributing to the pathogenesis of endothelial dysfunction [35]. Among others, BH4 and its derivatives have been implicated in various diseases, including phenylketonuria [36], neurological disorders [37], cardiovascular disease [38], [39] and diabetes [40], which have been reviewed elsewhere [23]. Enzymatic deficiencies in the BH4 biosynthesis and regeneration pathways can also give rise to additional pteridine derivatives. For example, the spontaneous rearrangement of BH4-4a-carbinolamine to a 7-substituted dihydrobiopterin has been reported in patients with low pterin-4a-carbinolamine dehydratase activity, leading to the formation of various 7-substituted pteridines that are excreted in urine [41], [42]. Meanwhile, non-enzymatic rearrangement of the unstable quinoid 6,7-dihydrobiopterin to 7,8-dihydrobiopterin can occur in the absence of 6,7-dihydropteridine reductase, where BH4 can be regenerated from 7,8-dihydrobiopterin via dihydrofolate reductase [43]. Finally, sepiapterin, a stereoisomer of biopterin, is hypothesized to result from the non-enzymatic rearrangement of unstable BH4 intermediates, possibly through alternative BH4 biosynthetic pathways, with elevated levels having been reported in patients with compromised sepiapterin reductase activity [44]. However, the biological functions and biosynthetic pathways for many of these peripheral pteridine derivatives remain to be established.
Neopterin is produced selectively in monocytes, macrophages, dendritic cells and endothelial cells following activation by the cytokines, interferon gamma (IFN-γ), and to lesser extents, interferons alpha (IFN-α) and beta (IFN-β) released by T lymphocytes and natural killer cells [45]. In addition, tumor necrosis factor-α (TNF-α) has been shown to amplify the stimulatory effects of IFN-γ on neopterin production [46]. The mechanism by which neopterin biosynthesis occurs in these cells is now understood to be the result of activation of GTP cyclohydrolase I by the aforementioned cytokines. Similar to the BH4 biosynthetic pathway, GTP cyclohydrolase I catalyzes the formation of 7,8-dihydroneopterin triphosphate from GTP. However, low physiological activity of 6-pyruvol-5,6,7,8-tetrahydropterin synthase, the next enzyme in the BH4 biosynthetic pathway, in monocytes and macrophages results in the accumulation of 7,8-dihydroneopterin triphosphate [46], [47]. Nonspecific oxidation and side chain cleavage further lead to the formation of neopterin and 7,8-dihydro neopterin in an approximate 1:2 ratio in urine, serum and cerebrospinal fluid [45], [48], [49]. Hence, the release of neopterin and its derivatives in response to cytokines have made them useful indicators of activated cell-mediated immunity. As a result, neopterin has been studied extensively as a disease biomarker for a wide array of medical disorders, including bacterial and viral infections, malignancy, autoimmune diseases and organ transplant failures, among others. Neopterin is frequently measured in urine given its constant excretion into urine via the kidneys and its relatively short half-life of 90 min in the circulatory system [50].
In addition to biopterin and neopterin derivatives, there are many other pteridines and related lumazines that have been reported in humans, but whose biological function and biosynthetic pathways remain to be established [7]. For example, a secondary entry point to the pteridine biosynthetic pathway involves the degradation of folates containing a pteridine moiety. In their seminal study, Fukushima and Shiota used radiographic tracers in Chinese hamster ovary cells to demonstrate that 6-hydroxymethylpterin and 6-carboxypterin were primarily derived from folates, as opposed to GTP, whereas pterin and its subsequent conversion to isoxanthopterin had mixed origins [51]. More recently, Burton and co-workers extended this work by examining the effects of folate supplementation on urinary levels of 6-biopterin, pterin, neopterin, xanthopterin, isoxanthopterin and 6-carboxypterin in healthy individuals who were not previously taking folate supplements [52]. The study findings were generally in agreement with the Fukushima study, with increased levels of pterin and 6-carboxypterin and decreased levels of 6-biopterin, neopterin and xanthopterin being reported. Moreover, the observation that GTP-derived pteridine levels decreased following folate supplementation suggests possible pathway crosstalk between the GTP and folate entry points. However, current understanding of the folate-derived pteridine biosynthetic pathway is limited mainly to observations of folate degradation in aqueous solutions [53], [54], [55], [56]. Namely, photocatalyzed dissociation of fully oxidized folic acid yields the highly reactive aldehyde, 6-formylpterin, which may be subsequently oxidized, either photocatalytically or spontaneously, to 6-carboxypterin and pterin [53]. Given that most biological folates exist in a reduced or semi-reduced state, Dántola and co-workers demonstrated that 7,8-dihydrofolic acid slowly oxidizes to 7,8-dihydroxanthopterin and, to a lesser extent, 6-formyl-7,8-dihydropterin [56]. In this regard, 7,8-dihydropterins bearing a CHOH- group at C(6), including 7,8-dihydroneopterin and 7,8-dihydrobiopterin, preferentially form 7,8-dihydroxanthopterin in the presence of oxygen. Notably, 7,8-dihydroxanthopterin is stable with a half-life of 1400 h at neutral pH solutions at room temperature [56]. The compound 7,8-dihydroxanthopterin has also been shown to be a major byproduct of the reaction of 7,8-dihydropterins with hydrogen peroxide under physiological conditions [57]. Meanwhile, the biosynthesis of xanthopterin and isoxanthopterin appears to be mediated by xanthine dehydrogenase which catalyzes the hydration of 7,8-dihydropterin and pterin, respectively [58]. The recent discovery and characterization of orphaned enzymes, such as isoxanthopterin deaminase and pterin deaminase, thought to be responsible for the biosynthesis and regulation of many peripheral pteridines may also provide researchers with new insights into their function in human health and disease [59], [60]. Current understanding on the pteridine metabolic pathway has been summarized in Figure 1.
![Figure 1: Proposed metabolic pathway of unconjugated pteridines from guanosine triphosphate and folic acid. Adapted with permission from Burton et al. [52]. Pteridines denoted with a *have been detected in urine.GTP, guanosine triphosphate; GTPCH I, guanosine triphosphate cyclohydrolase I; 7,8-NH2, 7,8-dihydroneopterin; 6-FPH2, 6-formyl-7,8-dihydropterin; PTPS, 6-pyruvoyl-tetrahydrobiopterin synthase; PPH4, 6-pyruvoyl-tetrahydrobiopterin; PH2, 7,8-dihydropterin; BH4, 5,6,7,8-tetrahydrobiopterin; 7,8-BH2, 7,8-dihydrobiopterin; XanH2, 7,8-dihydroxanthopterin; Xan, xanthopterin; PCD, 4a-hydroxytetrahydrobiopterin dehydratase; DHPR, 6,7-dihydropteridine reductase; AAAH, aromatic amino acid hydroxylase; AGMO, alkylglycerol monooxygenase; NOS, nitric oxide synthase; XDH, xanthine dehydrogenase.](/document/doi/10.1515/pterid-2016-0013/asset/graphic/j_pterid-2016-0013_fig_001.jpg)
Proposed metabolic pathway of unconjugated pteridines from guanosine triphosphate and folic acid. Adapted with permission from Burton et al. [52]. Pteridines denoted with a *have been detected in urine.
GTP, guanosine triphosphate; GTPCH I, guanosine triphosphate cyclohydrolase I; 7,8-NH2, 7,8-dihydroneopterin; 6-FPH2, 6-formyl-7,8-dihydropterin; PTPS, 6-pyruvoyl-tetrahydrobiopterin synthase; PPH4, 6-pyruvoyl-tetrahydrobiopterin; PH2, 7,8-dihydropterin; BH4, 5,6,7,8-tetrahydrobiopterin; 7,8-BH2, 7,8-dihydrobiopterin; XanH2, 7,8-dihydroxanthopterin; Xan, xanthopterin; PCD, 4a-hydroxytetrahydrobiopterin dehydratase; DHPR, 6,7-dihydropteridine reductase; AAAH, aromatic amino acid hydroxylase; AGMO, alkylglycerol monooxygenase; NOS, nitric oxide synthase; XDH, xanthine dehydrogenase.
Finally, new evidence for the relationship of folate metabolism with cancer, as well as the physicochemical properties of pteridine derivatives, has provided potential new roles of peripheral pteridines in disease. Briefly, urinary levels of various pteridines, including xanthopterin, isoxanthopterin and 6-carboxypterin have been reported to be increased in primarily epithelial cancers [3], [7], [18], [20], [61]. Although several competing theories have been proposed to explain the elevated pteridine levels reported in cancer patients, basic research supporting these claims have remained sparse. For example, recent work on the role of folate metabolism in cancer has implicated overexpression of the folate transporter, FOLR1, as a possible source for pteridine accumulation in some cancers. Briefly, FOLR1 is a membrane-bound protein that actively transports lipophilic folates into the cytoplasm via receptor-mediated endocytosis [62], and is overexpressed in certain epithelial tumors, including ovarian, breast, brain and lung cancers [62], [63], [64], [65], [66], which have closely corresponded with clinical observations of altered levels of peripheral pteridines in urine [7]. Another mechanism by which pteridines can affect pathology is regulating cellular oxidative stress. Many aromatic pteridines have pro-oxidant properties with the capability to generate singlet oxygen and superoxide anion radicals that counteract the free radical scavenging activity of reduced pteridines derivatives [67], [68], [69], [70]. For example, the neuroprotective effects [71] and suppression of inducible nitric oxide synthase activity [72] caused by 6-formylpterin, a potent xanthine oxidase inhibitor [73], has been attributed to its reactivity toward NAD(P)H in the presence of oxygen [74]. Similarly, photosensitization of aromatic pteridines and lumazines has been shown to cause oxidative damage to DNA and nucleotides [75], [76], [77], folic acid [54] and tryptophan [78] in vitiligo through the production of reactive oxygen species. Finally, the recently described interactions between the pro-oxidant folate-derived and anti-oxidant GTP-derived pteridine biosynthetic pathways suggest that pteridine metabolism may be regulated as a means to modulate cellular oxidative stress [52]. Taken together, pteridines and their derivatives compose a chemically diverse panel of metabolites with broad pathophysiology in humans. For this reason, the search for pteridine biomarkers has grown in popularity in recent years, marked by new technological advances in their quantitative determination and clinical studies aiming to examine their epidemiology in disease.
Analytical techniques for quantitation of urinary pteridines
Progress in understanding the relationship between pteridine metabolism and disease is fundamentally limited by the applicability of available analytical methods for their quantitative determination in biological matrices. As early as 1972, Rembold and Gyure recognized that basic research on pteridines had been frustrated by analytical difficulties relating to the chemical lability and trace levels of pteridines [79]. More recently, the rapid development of new analytical techniques has resulted in greater ability to characterize a multitude of pteridine derivatives, improved separation of structural isomers, and new preparative techniques for the determination of pteridines in various oxidation states. Kośliński et al. [24] recently reviewed cancer biomarker applications of unconjugated pteridines with a brief review of the primarily chromatographic and electrophoretic techniques available at the time. More recently, Tomšíková and co-workers [80] have expanded this review with detailed discussions on the stability of pteridines in biological samples with an emphasis on preparative techniques for pteridine bioanalysis. Nevertheless, the past several years have witnessed several emerging trends, such as increased reliance on sensitive mass spectrometry techniques, simultaneous determination of closely related pteridine derivatives and ongoing discussions on the applicability of preparative techniques for pteridine oxidation. We will briefly review the most salient aspects of available analytical methods for pteridine quantitation in urine as well as emerging trends in this area.
Sample preparation considerations
Pteridine derivatives naturally occur in three oxidation states: aromatic, dihydro- (semi-reduced) and tetrahydro- (fully reduced). Given the instability of the reduced and semi-reduced pteridine forms, urine samples have been conventionally pretreated to either fully oxidize or reduce pteridine derivatives to a single oxidation state [24], [80]. Various oxidative and anti-oxidative pretreatments have been developed for this purpose, including triiodide (I2/I−), permanganate (MnO4−), manganese dioxide, UV irradiation and hydrogen peroxide, among others, about which Tomšíková and co-workers have given a detailed review [80]. However, it is becoming increasingly recognized that different oxidation states of the same pteridine have different biological functions and clinical applicability as disease biomarkers. Moreover, the determination of urinary pteridines in their native oxidation states presents additional analytical challenges, since individual oxidation states have differences in solubility, hydrophobicity, intrinsic fluorescence quantum yield, susceptibility to auto-oxidation, ionization efficiency and molecular mass, among other physicochemical properties on which analytical methods are dependent. Several recent analytical methods for pteridine determination have begun to explore the feasibility of characterizing pteridines in their native oxidation states. Girón and co-workers were the first to measure urinary pteridines in their native oxidation states with the development of a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method using dithiothreitol as a preservative for the determination of ten pteridine derivatives [81]. Burton et al. have recently extended this work to the simultaneous determination of 15 pteridine derivatives in urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and comparing the performance of various oxidative pretreatments [7]. The latter study concluded that commonly used oxidative pretreatments, namely, triiodide, permanganate and manganese dioxide inefficiently oxidize reduced pteridines in urine matrices, leading to the formation of non-pteridinic byproducts and showing dependence on urine concentration-dilution. Since fluorimetric approaches are limited by the weak intrinisic fluorescence of reduced pteridine derivatives [80], [82], detection with mass spectrometry may be more favorable for the determination of native pteridines. Nevertheless, the clinical significance of measuring urinary pteridines in their native oxidation states, in which reduced species may be spontaneously oxidized in bladder urine, remains disputed and requires further investigation [83], [84], [85], [86].
In addition to (anti-)oxidative pretreatments, determination of urinary pteridines involves several special considerations with respect to sample collection and storage conditions. These considerations are related to the physicochemical properties of pteridines and their derivatives, which include trace biological concentrations and sensitivity to light, temperature and oxygen [4], [24]. For example, urine specimens must be maintained under dark conditions owing to the photosensitivity of pteridines and lumazines [82]. Moreover, storage temperature conditions for urine specimens vary considerably in the literature ranging from −70°C to 20°C [80] while the thermal stability of urinary pteridines with respect to time can vary from several months to several days at −20°C and from several hours to several days at 20°C. The apparent variability in reported thermal stability in the literature may be attributed to the lack of detailed studies on the subject as well as differences in stability experiment design. For example, Gibbons et al. reported relatively short half-lives for pteridines in freshly collected urine specimens at selected temperatures [87] compared with Tomšíková et al. who evaluated pteridine stability in solvent standards [86]. In addition, information on the thermal stability of pteridine derivatives in urine is generally restricted to several select compounds, such as biopterin and neopterin [80]. Given the increased attention to the determination of urinary pteridines in their native oxidation states, characterization of the thermal stability of these compounds in urine matrices is urgently needed. In addition, Burton et al. reported the gradual accumulation of 7,8-dihydroxanthopterin under prolonged storage at −80°C accompanied by freeze-thaw cycling [7]. The authors attributed this phenomenon to the non-specific oxidation and hydration of 7,8-dihydropteridines to 7,8-dihydroxathopterin [56]. In addition, Burton et al. have assessed the stability of urinary pteridines with respect to freeze-thaw cycling, reporting no significant losses following three successive freeze-thaw cycles between −80°C and room temperature [61]. A comprehensive review of pteridine stability in urine, as well as other biological matrices, is available [80].
Finally, urine specimens must be filtered and either diluted or pre-concentrated for compatibility with the selected analytical technique. Filtration is needed to remove residual proteins, exfoliated cells, cell debris and other sediments from urine. The formation of uncharacterized precipitates in urine can occur following sample collection, and this sedimentation process can be accelerated by freezing the sample. In healthy individuals, freezer-induced sediment mostly comprises calcium oxalate dehydrate crystals and entrapped proteins, such as uromodulin, albumin and bikunin [88]. Although entrapment of urinary pteridines has been reported to be minimal [61], these sediments can clog chromatographic columns [89], contaminate mass spectrometers [90] and contribute to baseline noise in spectrophotometric methods [3], [87]. In this regard, urine specimens have been conventionally filtered with either 0.45 μm or 0.22 μm nylon filters for quantitative determination of urinary pteridines in recently developed methods and clinical applications [3], [7], [52], [61], [87], [90], [91], [92], [93], [94], [95]. Urine samples can then be either diluted or pre-concentrated according to the biological levels of the selected pteridine analytes and sensitivity of the selected analytical technique, with dilution being preferred in the majority of cited works. Capillary electrophoresis separations are uniquely amenable to direct injection of urine samples; however, their hyphenation with laser-induced fluorescence detection modes requires oxidative pretreatments after which urine samples have been diluted up to three-fold [3], [87]. Meanwhile, dilution factors ranging from 10-fold to 250-fold have been used in a variety of liquid chromatography methods [7], [81], [90], [95]. In contrast, pre-concentration methods, such as solid-phase extraction, have been rarely reported for the determination of urinary pteridines in recent years. Although trace levels of urinary pteridines generally preclude the direct applicability of UV detectors, the increased prevalence of ultra-sensitive fluorescence and mass spectrometry detectors appears to have diminished the need for pre-concentration methods. Tomšíková and co-workers recently reported the development of a solid-phase extraction-ultra-high-performance liquid chromatography-fluorescence detection (SPE-UHPLC-FD) method for urinary levels of neopterin, 7,8-dihydroneopterin, biopterin and 7,8-dihydrobiopterin in which solid-phase extraction was used as a sample cleanup method in contrast to filtration [86]. The study authors examined three SPE sorbents, including the hydrophilic-lipophilic-balanced polymer phase Oasis HLB, the porous graphite carbon HyperSep Hypercarb and mixed-mode sorbent DSC-MCAX sorbent. Optimal recoveries were achieved with the DSC-MCAX sorbent using 20-fold diluted urine in 1% (w/v) dithiothreitol, washed with 1 mL water and 1 mL 1% acetonitrile in water, and finally eluted with 1 mL of acetonitrile and ammonia solution (75:25, v/v) in 1% (w/v) dithiothreitol and ranged from 74% to 122% in spiked urine samples. Given these values and the lack of contrary data that filtration adversely adsorbs urinary pteridines, the specific advantages gained by using this SPE procedure remain to be established. Finally, Tomandl and co-workers have reported using acid hydrolysis to urine samples in order to increase levels of oncopterin, a putative cancer biomarker [96], [97].
Current hyphenated techniques
Capillary electrophoresis-laser-induced fluorescence (CE-LIF)
Capillary electrophoresis is a high-resolution separation technique for the analysis of polar-ionic compounds that can be further hyphenated with laser-induced fluorescence for ultra-sensitive quantitation of fluorescent analytes. Capillary electrophoresis can provide several distinct advantages over liquid chromatography in applications involving the determination of urinary metabolites, such as reduced sample volumes, suitability to direct injection and a variety of separation modes. However, its applicability to urinary metabolite quantitation has been limited by poor reproducibility related to undesirable matrix and analyte adsorption on the capillary walls and sample matrix variability on which some injection techniques are dependent [98]. Nevertheless, several CE methods have been reported for the analysis of urinary pteridines. To summarize Kośliński et al. [24], Cha et al. initially developed a high-performance capillary electrophoresis-UV absorbance method for the determination of seven pteridines in urine, in which the sensitivity of the UV absorbance approach was determined to be unsuitable for the low levels of pteridines present in urine [99]. Han et al. extended this work by hyphenating capillary electrophoresis with laser-induced fluorescence, enabling successful detection [82]. As discussed by Han et al. one of the contributing factors to the poor reproducibility of CE techniques is the selection of the injection technique used, where Han et al. utilized electrokinetic injections that were influenced by the ionic strength of the samples. Gibbons et al. more recently built upon this work by developing specialized in-house CE-LIF instrumentation for the determination of urinary pteridines incorporating a more powerful laser source to enhance sensitivity to the picomolar regime and gravimetric injection techniques to improve reproducibility to clinically acceptable levels [87]. The Gibbons method additionally had a reported run time of 20 min, which is comparable to that of modern liquid chromatography methods [1], [81], [95], [100]. Despite the reported clinical applicability of these CE-LIF techniques to pteridine biomarkers [3], the development and application of new CE-based methods in recent years has been limited. Given the poor selectivity of spectrophotometric techniques, such as laser-induced fluorescence, the hyphenation of CE with mass spectrometry (CE-MS) represents a promising research direction. In this regard, the recent introduction of new CE-MS interface designs and advanced separation modes have overcome many of the early challenges associated with its application to urinary metabolites and is expected to contribute to urinary pteridine research in the near future [101].
High-performance liquid chromatography-fluorescence detection (HPLC-FD)
As previously mentioned, the low pathophysiological levels of pteridines present in urine generally precludes their detection using conventional UV absorbance techniques. Analogous to the development of CE-based methods, modern high-performance liquid chromatography methods for pteridine quantitation have begun to employ fluorescence detection as a means to exploit the intrinsic fluorescence displayed by many pteridine derivatives. Similar to CE-LIF methods, the reduced fluorescence quantum yields of the dihydro- and tetrahydro-pteridines, compared with their aromatic counterparts, generally require application of an oxidative process for best sensitivity. Nevertheless, in the past several years the accessibility of HPLC-FD compared with that of HPLC-MS(/MS) makes it an attractive platform for clinical biomarker screening applications, as evidenced by the development of several HPLC-FD methods for urinary pteridines. For example, Cañada-Cañada et al. reported an innovative online UV-photoirradiation method in conjunction with a fast scanning fluorimetric detector (FSFD) to enable post-column with the mobile photoderivatization for the determination of four urinary pteridines [102]. De Llanos et al. extended this work by examining a total of 11 pteridines and lumazines, in addition to urinary creatinine, and comparing the oxidative efficiency of common pretreatments [93]. The analytes were separated with a Zorbax Exclipse XDB-C18 analytical column, phase comprising 15 mmol/L Tris-HCL buffer at pH 6.1 and 6.4 under a gradient program, and a flow rate of 1.0 mL/min. The performance characteristics of the method were remarkable with comparable detection limits to CE-LIF (<7 ng/mL) and excellent reproducibility (<7% RSD), although method applicability was limited by the relatively long run time of 39 min. De Llanos et al. [91] and Culzoni et al. [92] also explored the feasibility of using second-order data obtained from the FSFD using multivariate curve resolution -alternating least squares algorithm modelling strategies. The advantage of utilizing the second-order data lies in its improved capacity to separate analytes from co-eluting spectral interferences, enhancing the selectivity of the HPLC-FD method. More recently, Guibal et al. utilized post-column coulometric oxidation to detect all forms of neopterin and biopterin in cerebrospinal fluid [103]. Nováková et al. examined the retention characteristics of bridged ethyl hybrid (BEH) and BEH amide hydrophilic interaction chromatography (HILIC) stationary phases using UHPLC-FD and UHPLC-MS techniques [104]. Kośliński et al. extended this work by comparing the retention capabilities of HILIC and conventional reversed-phase (RP) stationary phases for pteridine separation when hyphenated to fluorescence detection [95]. Briefly, the LiChrospher C(8) HILIC column afforded superior separation of urinary pteridines without compromising analytical sensitivity and reproducibility. Tornero et al. have recently studied the determination of pteridine derivatives in serum with HPLC-FD incorporating an acid precipitation step and clean-up process using an Isolute ENV+ (hydroxylated polystyrene-divinybenzene copolymer) cartridge [1]. This novel preparative step carries potential implications for the determination of urinary pteridines in samples with abnormal levels of proteins that arises in patients with renal dysfunction. In summary, determination of urinary pteridines with HPLC-FD is considered to be highly accessible, robust and applicable to clinical screening of pteridine biomarkers. Similar to CE-LIF, the technique is fundamentally limited by the poor selectivity afforded by spectrophotometric techniques in complex biological matrices, but new advances, such as the utilization of second-order spectral data are well positioned to address this significant challenge.
High-performance liquid chromatography-mass spectrometry (HPLC-MS)
The recent introduction of high-performance liquid chromatography-mass spectrometry methods represents a major breakthrough for the quantitative determination of pteridines in biological matrices. Mass spectrometry is well-suited for the analysis of polar urinary metabolites, which may be readily ionized using conventional ionization techniques, such as electron ionization (EI), atmospheric pressure-chemical ionization (APCI) and electrospray ionization (ESI), to provide extreme sensitivity comparable or superior to laser-induced fluorescence. Nevertheless, mass spectrometry alone has limited resolving power toward isobaric compounds, such as stereoisomers and structural isomers that are prevalent among pteridine derivatives (e.g. neopterin and monapterin, xanthopterin and isoxanthopterin), requiring either tandem mass spectrometry, specialized chemical derivatization techniques or hyphenated separation techniques. As a result, mass spectrometry methods for urinary pteridines must consider several factors. First, the ionization process should be compatible with the physicochemical properties of the analytes. In this regard, ESI ionization processes have been preferred for the analysis of urinary pteridines [7], [81], [90], [100], [105], [106], [107], [108], [109]. With the exception of the method developed by Allegri et al. in which negative-mode ESI was selected for greater sensitivity of neopterin and biopterin [100], positive-mode ESI has been employed for the quantitation of urinary pteridines. Moreover, Burton et al. have studied the sodiation and ammoniation of pteridine derivatives in positive-mode ESI conditions resulting from use of ammonium hydroxide to enhance the solubility of pteridine derivatives and ubiquitous sodium [7], [90]. These molecule adducts can contribute to decreased sensitivity and require complicated ion summing procedures. The authors found that pteridine derivatives with electron-withdrawing substituents, such as xanthopterin, favored the formation of ammoniated adducts while unsubstituted pteridine derivatives, such as pterin, favored the formation of sodiated adducts. Dissolution of pteridine derivatives with sodium hydroxide resulted in over 80% of pterin forming sodiated adducts, whereas dissolution with ammonium hydroxide resulted in approximately 30% adduction of xanthopterin and 6-carboxypterin [90]. However, addition of 0.1 mmol/L ammonium oxalate as a sodium scavenger [90] and higher declustering potentials in the ion source [7] can minimize adduction of pteridines in urine matrices. Burton et al. have also reported [M+15]+ and [M+32]+ molecular ions in the mass spectrum of 6-formylpterin in the presence of methanol, which were attributed to gas-phase aldol reactions [7]. In addition to molecular adduction, the possibility of in-source oxidation of labile pteridine derivatives has been recently considered. Van Daele and co-workers reported thermally facilitated in-source oxidation of 6-hydroxymethyl-7,8-dihdyropterin and 7,8-dihydroxanthopterin [106]. In comparison, Burton et al. reported in-source oxidation of 6-formylpterin to 6-carboxypterin, which could be mitigated with co-infusion of 100 μmol/L ascorbic acid or dithiothreitol and minimal in-source oxidation of 7,8-dihydroxanthopterin under similar conditions [7]. First, further study will be needed to determine the impact of in-source oxidation on urinary pteridines. Second, ion suppression related to the eluent matrix cannot be predicted or controlled in the analysis of urine specimens with ESI-MS methods given their sensitivity toward charge competition in the ion source. The most practical solution to this problem is the utilization of stable isotope-labeled internal standards. In this regard, Arning et al. used 15N-stable isotopes of 5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin and neopterin to determination in cerebrospinal fluid [108]. Stable isotope-labeled internal standards are advantageous given their co-elution and matching basicity with corresponding analytes. Unfortunately, the accessibility of stable isotope-labeled internal standards, and particularly with respect to pteridine derivatives, remains prohibitive. As a result, the utilization of a single, structurally related pteridine derivative that has no or little endogenous levels, such as 6,7-dimethylpterin, 6-methylpterin or aminopterin, as an internal standard has been reported in some methods [81], [106]. However, this approach cannot account for localized ion suppression at different elution times and cannot compensate for differences in compound basicity. In response, Allegri et al. infused individual pteridine standards with a urine sample blank to measure localized matrix effects, although this approach is considered laborious [100]. Further study on approaches to estimate ion suppression in ESI-MS methods in the determination of urinary pteridines is urgently needed. Finally, high-resolution or tandem mass spectrometers provide several advantages over conventional mass spectrometers including enhanced selectivity, sensitivity and capacity to differentiate isobaric compounds, such as 6,7-dimethylpterin and 6-formylpterin (both m/z 192.1). In this regard, several recent HPLC-MS/MS methods have been developed for the quantitation of pteridines in biological samples [7], [90], [100], [105], [106], [108], [109]. The robustness of these new methods has led to enhanced multiplexing capabilities allowing for a greater number of pteridine derivatives to be quantified simultaneously. Recently, Burton et al. reported the quantitative determination of 15 pteridine derivatives in urine, opening the door to what the authors referred to as quantitative “pterinomics” [7]. A summary of the analytical capabilities of the methods reviewed here is presented in Table 1.
Summarized information on recently developed hyphenated techniques for pteridine quantitation.
| Technique | Pteridines | Oxidative pretreatment | Run time, min | Limits of detection, nmol/L | Reference |
|---|---|---|---|---|---|
| CE-LIF | PTE, XAN, ISO, NEO, 6-BIO, 6-CAP, 6,7-DMP, 6-HMP | Alkaline I3− | 20 | 0.25–0.50 | Gibbons et al. [87] |
| HILIC-FD (MS) | NEO, NH2, BIO, BH2 | None | 8 (4) | Not given | Nováková et al. [104] |
| HPLC-FD | PTE, XAN, ISO, NEO, 6-BIO, 7-BIO, MNP, 6-CAP, 6-HMP, LUM, 6-HLUM, 7-HLUM, BLUM | Alkaline I3− and KMnO4 | 39 | 0.8–27 | De Llanos et al. [93] |
| HPLC-FD | PTE, NEO, 6-BIO; XAN, ISO | None | 16 | 0.05–0.5 | Culzoni et al. [92] |
| HPLC-MS/MS | PTE, ISO, 6-BIO, 7-BIO, NEO, MNP | Acidic MnO2 | 19 | 0.03–2 | Allegri et al. [100] |
| HPLC-MS | PTE, XAN, ISO, NEO, MNP, NH2, 6-BIO, BH2, 6-HMP, 6-MP, 6-CAP | None | 30 | 10–300 | Girón et al. [81] |
| HPLC-MS/MS | PTE, XAN, ISO, NEO, 6-BIO, 6-CAP, 6,7-DMP, 6-HMP | Alkaline I3− | 7.5 | 0.1–2.5 | Burton et al. [90] |
| HILIC-FD | 6-BIO, BH2, BH4, NEO, NH2 | None | 16 | 0.9–1.8 | Guibal et al. [103] |
| HPLC-FD | PTE, XAN, ISO, NEO, 6-BIO, 6-CAP, 6,7-DMP, 6-HMP | Alkaline I3− and KMnO4 | 25 | 0.2–20 | Kośliński et al. [95] |
| HPLC-MS/MS | BH4, BH2, NEO, SEP | None | 10 | 1 | Arning and Bottiglieri [108] |
| HPLC-FD | PTE, XAN, ISO, NEO, MNP, 6-BIO, 7-BIO, 6-CAP, 6-HMP | Acidic I3− | 29 | 0.2–2 | Tornero et al. [1] |
| SPE-HILIC-FD | NEO, NH2, BIO, BH2 | None | 8+SPE | 4–100 | Tomšíková et al. [86] |
| HPLC-MS/MS | Quantitative: PTE, XAN, ISO, NEO, 6-BIO, 6-CAP, 6,7-DMP, 6-HMP Semi-quantitative: LUM, PH2, PH4, 6-MP, 6-HLUM, 7-HLUM, XH2, 6-FOP, 6,7-DMH4P; BH2, BLUM, BH2, BH4, MNP, NH2, NH4 | None | 25 | 0.4–17 | Burton et al. [105] |
| UHPLC-MS/MS | PTE, XAN, XH2, NEO, NH2, 6-HMP, 6-HMDP, 6-FOP, 6-CAP | Mn2O5 | 8 | Not Given | Van Daele et al. [106] |
| HPLC-MS/MS | PTE, XAN, XH2, ISO, NEO, MNP, 6-BIO, SEP, 6-CAP, 6,7-DMP, 6-MP, 6-HMP, LUM, 6-HLUM, 7-HLUM | None | 7 | 0.1–3 | Burton et al. [7] |
| HILIC-MS/MS | PTE, XAN, ISO, 6,7-DMP, 6,7-DMH4P, 6-CAP, 6-BIO, BH2, BH4, SEP, NEO, NH2 | None | 20 | Not Given | Xiong and Liu [109] |
Adapted from Burton et al. [7]. PTE, pterin; XAN, xanthopterin; ISO, isoxanthopterin; NEO, neopterin; 6-BIO, 6-biopterin; 6-CAP, 6-carboxypterin; 6,7-DMP, 6,7-dimethylpterin; 6-HMP, 6-hydroxmethylpterin; 7-BIO, 7-biopterin; MNP, monapterin; LUM, lumazine; 6-HLUM, 6-hydroxylumazine; 7-HLUM, 7-hydroxylumazine; BLUM, biolumazine; NH2, 7,8-dihydroneopterin; BH2, 7,8-dihydrobiopterin; 6-MP, 6-methylpterin; BH4, tetrahydrobiopterin; XH2, 7,8-dihydroxanthopterin; PH2, dihydropterin; PH4, tetrahydropterin; 6,7-DMH4P, 6,7-dimethyltetrahydrobiopterin; NH4, tetrahydroneopterin; 6-HMDP, 6-hydroxymethyldihydropterin; 6-FOP, 6-formylpterin; SEP, sepiapterin.
Clinical study of pteridine biomarkers in urine
In recent years, a number of clinical studies have been performed to investigate the prevalence of urinary pteridines in association with various diseases. Briefly, pteridine fingerprinting or “pterinomics”, has been most aggressively applied to the area of cancer diagnostics and is discussed in great detail here. In addition, the role of urinary neopterin as an inflammatory biomarker has been widely studied in different diseases. We have therefore attempted to compile a non-exhaustive list of clinical studies on the role of urinary pteridines as disease biomarkers performed in the past 5 years. We have additionally restricted our search to studies with sample sizes of at least 25 participants in order to facilitate a meaningful review of the clinical significance of urinary pteridines as disease biomarkers.
Cancer
For the past several decades, urinary pteridines have been studied extensively as putative biomarkers for early cancer detection and diagnosis following early reports of altered levels in cancer patients [18], [20]. Kośliński et al. recently reviewed these earlier clinical studies, concluding that pteridines presented a promising direction for cancer biomarker research and that further advances in analytical technologies would be needed to evaluate their significance in biological and clinical contexts [24]. Indeed, new advances in analytical methods have opened new areas of exploration for pteridine research, including new understanding on their epidemiology in cancer patients and the putative mechanisms by which pteridines may accumulate in cancer patients. A number of clinical studies have been recently conducted to profile urinary pteridines in cancer patients, which have been reviewed here and salient aspects of these clinical studies having been summarized in Table 2.
Summary of recent clinical studies on the prevalence of urinary pteridines in cancer.
| Cancer | Pteridines studied | Technique | Oxidation | Urine normalization | Sample size (cases/controls)a | Matched controls | Altered pteridines | Reference |
|---|---|---|---|---|---|---|---|---|
| Breast | NEO, 6-BIO, XAN, 6-HMP, PTE, ISO, 6,7-DMP, 6-CAP | CE-LIF | I2/I − | CRE | 29 (12/17) | No | ↑6-BIO, ↑6-HMP, ↑PTE, ↑ISO, ↑XAN, ↑6-CAP | Gamagedara et al. [3] |
| Breast | NEO | HPLC-FD | None | CRE | 456 (456/0) | N/A | ↑NEO | Kalábová et al. [110] |
| Breast | NEO, 6-BIO, XAN, 6-HMP, PTE, ISO, 6,7-DMP, 6-CAP | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | No | None | Burton et al. [90] |
| Breast | NEO, 6-BIO, XAN, 6-HMP, PTE, ISO, 6,7-DMP, 6-CAP | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | Yes | ↑XAN, ↑ISO | Burton et al. [61] |
| Thyroid | NEO | ELISA | None | CRE | 178 (69/109) | Yes | ↑NEO | Inancli et al. [111] |
| Ovarian | NEO, 6-BIO, PTE | HPLC-FD | MnO2 | CRE | 107 (75/32) | No | ↑NEO, ↑6-BIO, ↑PTE | Zvarik et al. [112] |
| Breast | PTE, XAN, XH2, ISO, NEO, MNP, 6-BIO, SEP, 6-CAP, 6,7-DMP, 6-MP, 6-HMP, LUM, 6-HLUM, 7-HLUM | HPLC-MS/MS | None | USG | 50 (25/25) | Yes | None | Burton et al. [7] |
| Bladder | NEO, 6-BIO, PTE, 6-CAP, ISO, XAN | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | No | ↓XAN, ↓ISO | Kośliński et al. [94] |
aFor simplification purposes, benign tumors have been treated numerically as controls. Readers are encouraged to refer to the cited literature for more information. CRE, creatinine; USG, urine specific gravity; PTE, pterin; XAN, xanthopterin; ISO, isoxanthopterin; NEO, neopterin; 6-BIO, 6-biopterin; 6-CAP, 6-carboxypterin; 6,7-DMP, 6,7-dimethylpterin; 6-HMP, 6-hydroxmethylpterin; MNP, monapterin; LUM, lumazine; 6-HLUM, 6-hydroxylumazine; 7-HLUM, 7-hydroxylumazine; 6-MP, 6-methylpterin; XH2, 7,8-dihydroxanthopterin; SEP, sepiapterin.
Breast cancer
In 2011, Gamagedara et al. measured eight urinary pteridines in 38 individuals with assorted cancers and in 17 healthy men and women using CE-LIF [3]. Urine samples were pretreated with triiodide in order to achieve optimal sensitivity for the CE-LIF method and urinary pteridine concentrations were adjusted to urinary creatinine to correct for urine concentration-dilution. The study reported elevated levels of 6-biopterin, 6-hydroxymethylpterin, pterin, xanthopterin and isoxanthopterin in cancer patients compared with the healthy controls. The greatest differences were observed between women with breast cancer (n=12) vs. healthy controls. As a result, the study has received a number of citations in recent years as evidence that urinary pteridines may serve as cancer biomarkers. However, several aspects of the clinical study design have substantially weakened the potential impacts of its findings. First, the statistical analyses may have been underpowered given the limited sample size used and the sample heterogeneity. Moreover, the case-controls were not age- or gender-matched, with the controls composed primarily of college-aged males. As later discussed by Burton et al. [61], [90], significant differences in urinary creatinine, which differs with age, gender, physical activity and other factors, likely contributed to the reported differences in urinary pteridine levels.
Burton et al. extended this work by examining urinary pteridines in matched urine samples collected from women with benign and aggressive breast cancers using HPLC-MS/MS [7], [61], [85], [90]. Using similar preparative procedures, Burton and co-workers reported no significant differences in urinary pteridines in a sample cohort comprising 12 women with pathologically verified breast cancer and 13 women with benign fibrocystic changes [90]. In addition, the authors compared the HPLC-MS/MS results with data collected using the CE-LIF technique as Gamagedara et al. [3]. This comparison indicated that xanthopterin and isoxanthopterin concentrations were significantly higher when measured with the CE-LIF technique than with the HPLC-MS/MS method. These differences were attributed to co-eluting spectral interferences in the CE-LIF technique. In order to further characterize the significance of altered creatinine levels, Burton et al. examined the association of urinary pteridines with breast cancer using urine specific gravity (USG) as an alternative adjustment factor for urine concentration-dilution in a cohort of 48 matched women with benign and aggressive breast cancers [61]. The study reported significantly elevated levels of xanthopterin and isoxanthopterin in the women with breast cancer when adjusted with USG whereas no significant differences were reported using urinary creatinine. In addition, the correlation between urinary pteridine concentrations and patient age was found to be negligible. More recently, Burton and co-workers have studied the association of 15 pteridine derivatives in their native oxidation states in a similar patient cohort using an expanded HPLC-MS/MS method [7]. No significant differences were reported between women with aggressive and benign breast cancers using this new technique, suggesting that the oxidation of reduced pteridine pools used in earlier studies may have contributed to previous reports of altered pteridine levels.
Finally, Kalábová et al. measured urinary neopterin in 456 patients with breast cancer using HPLC-FD to study its prognostic significance [110]. Briefly, increased levels of neopterin were reported in some patients and were generally associated with poor survival. Specifically, elevated neopterin levels were also reported among a subset of 55 patients with metastatic or recurrent breast cancers compared with other patients (224±203 μmmol/mmol creatinine vs. 176±177 μmmol/mmol creatinine) as well as patients who had previously received therapy (211±274 μmmol/mmol creatinine vs. 159±59 μmmol/mmol creatinine). Urinary neopterin levels did not correlate with breast cancer stage. Moreover, its prognostic significance differed with tumor histology, where increased urinary neopterin was more closely associated with progesterone receptor and estrogen receptor positive tumors as well as human epidermal growth factor receptor (HER)-2 tumors than triple negative tumors. The authors concluded that urinary neopterin is increased within a minority of breast carcinomas and may be a useful prognostic indicator for poor survival.
Colon cancer
Melichar et al. investigated the effects of patupilone therapy on urinary levels of neopterin in 17 men and women with metastatic colon cancers [113]. Urinary neopterin was measured with HPLC-FD and adjusted to urine concentration-dilution with urinary creatinine. The authors reported increased levels of urinary neopterin after initial screening and prior to start of therapy, suggesting an activated immune response to the metastatic cancers. The elevated levels reported in untreated patients supported similar findings made in patients with advanced colorectal cancers [114]. Treatment with patupilone further increased urinary neopterin levels, suggesting that patupilone therapy is capable of evoking an immune response for which urinary neopterin can serve as a useful therapeutic monitoring biomarker. However, further studies will be needed to validate these findings given the small sample size of the study, as well as differences in patupilone administration that may have contributed to the study findings.
Bladder cancer
Kośliński et al. recently examined urinary levels of neopterin, 6-biopterin, pterin, 6-carboxypterin, isoxanthopterin and xanthopterin in 81 patients with and without bladder cancer [94]. Urinary pteridines were measured using HPLC-FD with a triiodide oxidation pretreatment and creatinine adjustments. Slight increases in the levels of urinary xanthopterin and isoxanthopterin were reported in the bladder cancer patients compared with the controls. However, logistic regression models afforded non-significant associations with risk of bladder cancer, while the receiver-operator characteristic (ROC) curves derived from these models suggested weak biomarker performance with areas-under-the-curve less than 0.63. Similar to the Gamagedara et al. study [3], significant differences in patient age were reported between the study groups (mean age controls: 54.8±14 years; mean age cases: 70.1±12 years), which may have contributed to different levels of urinary creatinine between the two groups. However, descriptive statistics for creatinine levels were not published, so the contribution of creatinine to the reported differences in urinary pteridine levels cannot be ruled out. Further study will be needed to determine the generalizability of these findings.
Ovarian cancer
Elevated levels of urinary neopterin had been earlier reported in approximately 80% of women with ovarian cancers at time of diagnosis [21], [115]. Zvarik et al. have recently extended this work by examining the levels of neopterin, 6-biopterin and pterin in urine in women with benign and malignant ovarian cancers compared with healthy individuals. The clinical study comprised 36 women with malignant ovarian cancers, 39 women with benign ovarian tumors and 32 healthy individuals. Urinary pteridines were first pretreated with manganese dioxide and their concentrations were adjusted to urinary creatinine. The study reported significantly elevated levels of all three pteridines in patients with benign and malignant tumors compared with the healthy controls. Moreover, levels of urinary neopterin were significantly higher in malignant cases than compared with benign cases, suggesting that neopterin may have the capacity to distinguish disease aggressiveness. The study results are promising in the context of ovarian cancer detection and diagnosis. However, the practice of using manganese dioxide to oxidize pteridines has since been discouraged owing to its capacity to generate unfavorable byproducts including non-pteridinic compounds [7], [95]. Larger studies will be needed to examine the generalizability of these findings.
Other medical conditions
Neopterin as an atherosclerosis risk factor in cancer patients
Králíčková et al. studied the correlation between CD14+CD16+ monocytes, urinary neopterin, serum cholesterol, homocysteine, C-reactive protein, retinol and serum α-tocopherol in patients with breast cancer to determine whether history of breast cancer increases risk for atherosclerosis [116]. Urinary neopterin was measured in spot urine specimens taken from 33 women with histologically verified breast cancers using HPLC-FD. However, the study reported no correlation between CD14+CD16+ monocytes and urinary neopterin levels. The study authors suggested the small sample size may have contributed to the lack of correlation.
Neopterin as a biomarker for occupational exposure in operating rooms
Baydar et al. measured urinary neopterin concentrations in operating room personnel to determine whether neopterin correlated with occupational exposure in a typical operating room setting [117]. Urinary neopterin was measured with HPLC-FD and adjusted to urinary creatinine levels. A total of 40 men and women who worked in surgery and anesthesiology clinics and 30 healthy controls were studied. The authors reported higher levels of urinary neopterin in the operating room personnel than in the healthy controls (controls: 85±16 μmol/mol creatinine; operating room personnel: 151±39 μmol/mol creatinine, p<0.05). The authors concluded that the higher levels of urinary neopterin were indicative of occupational exposures to various infectious agents, low levels of anesthetic gases, cleaning supplies and chemical solvents.
Neopterin in Crohn’s disease and ulcerative colitis
Husain et al. recently studied fecal, serum, urinary levels of neopterin in patients with Crohn’s disease and ulcerative colitis to determine whether neopterin can serve as an index of disease activity [118]. A total of 70 patients with Crohn’s disease (33 clinically in remission, 37 active), 52 patients with ulcerative colitis (29 clinically in remission, 23 active) and 141 healthy controls were examined in this study. Although neither serum nor urinary levels of neopterin were associated with presence of disease, fecal concentrations were increased in both diseases. Moreover, fecal neopterin was increased in both active and inactive Crohn’s disease, whereas higher levels were reported for only clinically active ulcerative colitis.
Neopterin in chronic heart failure
Caruso et al. examined whether urinary levels of neopterin associated with left ventricular remodeling and brain natriuretic peptide in chronic heart failure patients [119]. Chronic heart failure involves a state of immune activation with persistent expression of pro-inflammatory cytokines that stimulate neopterin production [120]. As a result, neopterin is an active participant in cardiovascular diseases, with high serum levels being associated with higher rates of cardiac events in heart failure patients [121]. This study measured urinary neopterin in 98 patients with myocardial dysfunction secondary to ischemic or idiopathic dilated cardiomyopathy and 19 controls using HPLC-FD and creatinine adjustments. Significantly higher levels of urinary neopterin (mean: 0.183 vs. 0.120 μmol/mmol creatinine, p=0.001) were observed in chronic heart failure patients compared with the healthy controls. Moreover, neopterin levels were associated with left ventricular enlargement and interleukin-8 levels in chronic heart failure patients, suggesting that monocyte activation is involved with left ventricular remodeling.
Future perspectives and emerging challenges
The challenge of method variability
A variety of analytical techniques for the quantitative determination of pteridines derivatives in urine have been developed and applied to clinical studies as previously discussed. However, differences in analytical techniques, oxidative pretreatments and other sample preparation methods, as well as adjustments to urine concentration-dilution, can yield variable levels of pteridine derivatives in urine. In order to better illustrate this point, reference values for 15 urinary pteridines have been compiled from the clinical studies reviewed here and summarized in Table 3. Although some clinical studies have reported similar reference ranges using similar quantitative methods, reference ranges reported in other studies appear to differ substantially. For example, Kośliński et al. reported pteridine levels multiple orders of magnitude less than those reported in other studies [94]. Meanwhile, the ELISA method used by Inancli et al. was capable of detecting remarkably high concentrations of urinary neopterin (>1.5 μmol/mmol creatinine), although their mean values were consistent with other studies [111]. Similarly, Burton et al. compared the effect of oxidative pretreatments in which the application of oxidative pretreatments resulted in higher values of 6-biopterin, neopterin and xanthopterin as their reduced derivatives were converted by the oxidation process [7], [61]. In this way, future studies are strongly encouraged to compare their pteridine concentrations with known reference ranges in order to identify potential methodological bias. Similarly, technical standardizations will ensure better accuracy and reproducibility in clinical studies.
Reference values of urinary pteridines in different medical disorders as measured by various techniques, oxidative pretreatments and urine normalization methods.
| Medical disorder | Technique | Oxidative pretreatment | Urine normalization | Sample size (cases/controls) | Mean (range) cases, μmol/mmol CREa | Mean (range) controls, μmol/mmol CREa | Reference |
|---|---|---|---|---|---|---|---|
| Neopterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 1.14 | 0.24 | Gamagedara et al. [3] |
| Breast cancer | HPLC-FD | None | CRE | 33 (33/0) | 0.20 (0.10–0.52) | N/A | Králíčková et al. [116] |
| Myocardial dysfunction | HPLC-FD | None | CRE | 117 (98/19) | 0.18 (0.14–0.25) | 0.12 (0.10–0.21) | Caruso et al. [119] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.09–0.99) | (0.09–0.99) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 14.2 μmol/L | 9.6 μmol/L | Burton et al. [61] |
| Thyroid cancer | ELISA | None | CRE | 178 (69/109) | 0.149 (0.015–1.602) | 0.009 (0.003–0.079) | Inancli et al. [111] |
| Ovarian cancer | HPLC-FD | MnO2 | CRE | 107 (75/32) | 0.226 (0.163–0.330) | 0.056 (0.048–0.080) | Zvarik et al. [112] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.0007 (0.00002–0.005) | 0.0007 (0.00009–0.005) | Kośliński et al. [94] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.18–26.30 μmol/L) | (0.18–26.30 μmol/L) | Burton et al. [7] |
| 6-Biopterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 0.9 | 0.11 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.12–1.01) | (0.12–1.01) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 15.5 μmol/L | 10.0 μmol/L | Burton et al. [61] |
| Ovarian cancer | HPLC-FD | MnO2 | CRE | 107 (75/32) | 0.239 (0.187–0.321) | 0.096 (0.072–0.132) | Zvarik et al. [112] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.11–9.7 μmol/L) | (0.11–9.7 μmol/L) | Burton et al. [7] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.0006 (0.00004–0.006) | 0.0005 (0.00005– 0.002) | Kośliński et al. [94] |
| Pterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 0.6 | 0.175 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.014–0.633) | (0.014–0.633) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 2.9 μmol/L | 2.0 μmol/L | Burton et al. [61] |
| Ovarian cancer | HPLC-FD | MnO2 | CRE | 107 (75/32) | 0.056 (0.034–0.111) | 0.020 (0.008–0.040) | Zvarik et al. [112] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.15–2.34 μmol/L) | (0.15–2.34 μmol/L) | Burton et al. [7] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.0005 (0.00002–0.0025) | 0.0003 (0.00002–0.001) | Koslinski et al. [94] |
| Xanthopterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 5.6 | 1.1 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.126–1.434) | (0.126–1.434) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 9.1 μmol/L | 5.6 μmol/L | Burton et al. [61] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.32–6.00 μmol/L) | (0.32–6.00 μmol/L) | Burton et al. [7] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.0021 (0.00009–0.0016) | 0.0015 (0.00005–0.0074) | Koslinski et al. [94] |
| Isoxanthopterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 33.7 | 1.1 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.025–0.191) | (0.025–0.191) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 3.6 μmol/L | 1.0 μmol/L | Burton et al. [61] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.25–2.01 μmol/L) | (0.25–2.01 μmol/L) | Burton et al. [7] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.00150 (0.000040–0.01000) | 0.00020 (0.00001–0.00100) | Koslinski et al. [94] |
| 6-Hydroxymethylpterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 0.48 | 0.05 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | (0.025–0.191) | (0–0.160) | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | 4.73 μmol/L | 3.37 μmol/L | Burton et al. [61] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | N.D | N.D. | Burton et al. [7] |
| 6-Carboxypterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 0.254 | 0.125 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | N.D. | N.D. | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | N.D. | N.D. | Burton et al. [61] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | N.D. | N.D. | Burton et al. [7] |
| Bladder cancer | SPE-HPLC-FD | I2/I− | CRE | 81 (46/35) | 0.00027 (0.00001–0.00274) | 0.00024 (0.00006–0.00115) | Koslinski et al. [94] |
| 6,7-Dimethylpterin | |||||||
| Assorted cancers | CE-LIF | I2/I− | CRE | 29 (12/17) | 0.741 | 0.028 | Gamagedara et al. [3] |
| Breast cancer | HPLC-MS/MS | I2/I− | CRE | 25 (12/13) | N.D. | N.D. | Burton et al. [90] |
| Breast cancer | HPLC-MS/MS | I2/I− | USG | 48 (21/27) | N.D. | N.D. | Burton et al. [61] |
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | N.D. | N.D. | Burton et al. [7] |
| Monapterin | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.05–2.40 μmol/L) | (0.05–2.40 μmol/L) | Burton et al. [7] |
| 7,8-Dihydroxanthopterin | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (5–112 μmol/L) | (5–112 μmol/L) | Burton et al. [7] |
| 6-Hydroxylumazine | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | N.D. | N.D. | Burton et al. [7] |
| Lumazine | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.19–1.00 μmol/L) | (0.19–1.00 μmol/L) | Burton et al. [7] |
| 7-Hydroxylumazine | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.067–0.465 μmol/L) | (0.067–0.465 μmol/L) | Burton et al. [7] |
| 6-Methylpterin | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | N.D. | N.D. | Burton et al. [7] |
| Sepiapterin | |||||||
| Breast cancer | HPLC-MS/MS | None | USG | 50 (25/25) | (0.022–4.500 μmol/L) | (0.022–4.500 μmol/L) | Burton et al. [7] |
aDefault units are in μmol pteridine/mmol creatinine. Published values using different creatinine units (e.g. μmol pteridine/mol creatinine) were converted to the default format. In comparison, normalization of pteridine concentrations by USG affords molar units, and these units have been provided where applicable. N.D., not detected; CRE, creatinine; USG, urine specific gravity.
Limitations in clinical study design
The development and application of urinary pteridines as disease biomarkers, with the notable exceptions of neopterin and BH4 derivatives, remain at a very early stage. As a result, the majority of clinical studies reviewed here have been primarily exploratory in nature, characterized by having limited sample sizes and failing to control for potential confounding factors. For example, urinary pteridines must be adjusted to urine concentration-dilution in order to correct for patient hydration status, renal function and time since last urination. However, previous work has utilized inconsistent or inadequate normalization factors, as is evident from Table 3. Briefly, urinary creatinine is widely used for this purpose owing to its presumed constant production [122] and excretion [123]. However, this assumption is fundamentally flawed, since creatinine is synthesized primarily in muscle tissue as the breakdown product of creatine phosphate, resulting in its association with a number of clinicopathological factors including age, race, gender [124], [125], [126], physical activity, muscle mass [127] and diet [128], [129], [130]. Hence, urinary creatinine levels can differ significantly across study groups if these factors are not well controlled. For this reason, researchers are encouraged to report urinary creatinine levels for individual study groups and match controls where possible.
In comparison, USG is another adjustment factor that represents the ratio between the density of urine and pure water [131]. As a bulk property of urine, USG may be more robust than urinary creatinine for adjusting urinary metabolite concentrations. In this regard, several recent studies have reported improved performance of putative biomarkers in case-control studies using this approach [61], [132]. Burton et al. demonstrated significantly elevated levels of xanthopterin and isoxanthopterin in women with breast cancer using USG compared with urinary creatinine [61]. However, USG is not a true measure of solute concentration and is influenced by the mass distribution of the solution. Some medical disorders, such as proteinuria, can result in elevated values [133], [134], while others, like ketoacidosis in patients suffering from diabetes mellitus, can decrease values [135], [136]. Urine osmolality can provide an accurate measurement of solute concentration and is considered the “gold standard” for adjusting urinary biomarkers, but requires specialized equipment. In summary, special care must be taken in the design of clinical studies for urinary metabolites with respect to sample size, patient factors and method selection.
Biological variation of urinary pteridines
Finally, the determination of urinary metabolites presents special challenges in the interpretation of their clinical significance. This problem tends to be exacerbated in spot urine specimens, in which sample collection time, in relation to different biological processes, can alter urinary metabolite levels. For example, Slupsky et al. studied the changes of the urine metabolome in relation to diurnal variations showing a pronounced effect [137]. Similarly, Giskeødegård et al. demonstrated that sleep deprivation resulting in disrupted circadian rhythms, affected primary and secondary metabolite synthesis and excretion into urine [138]. In the context of urinary pteridines, Burton et al. recently studied the effects of sample collection time, in relation to circadian rhythms, as well as folate supplementation on urinary pteridine levels [52]. Significant changes in the levels of urinary 6-biopterin, pterin, neopterin, xanthopterin and isoxanthopterin were reported throughout the day, with the highest and the most consistent levels occurring in the morning. Moreover, the within-day and between-day variation of urinary pteridines, within the same individual, was substantial, averaging approximately 30% for both values. Folate supplementation at the daily recommended level in healthy individuals who were not previously taking folate supplements similarly resulted in significant changes in urinary pteridine levels following 2 weeks of supplementation, ranging from −32% baseline levels for xanthopterin to +27% baseline values for pterin. In this way, natural variations in urinary pteridine excretion represent potential confounding factors in the interpretation of their clinical significance as disease biomarkers, and should be controlled for where possible. We anticipate the introduction of new strategies to measure and control biological variation for urinary metabolites in the near future as this challenge becomes more prominent.
Concluding remarks
Pteridines and their derivatives broadly function as intermediates in the metabolism of several vitamins and cofactors, and their relevance to disease has resulted in new efforts to study their clinical significance as disease biomarkers. Recent analytical advances, including the emergence of sensitive mass spectrometry techniques, new methods for quantifying pteridines in their native oxidation states and increased multiplexing capacity for simultaneous determination of many pteridine derivatives, have enabled researchers to study urinary pteridines as disease biomarkers with greater ease and accuracy. As a result, pteridine derivatives are being studied increasingly as putative cancer biomarkers with promising results being reported from exploratory studies. In addition, the role of urinary neopterin as a universal biomarker for immune system activation is being explored in new diseases where it is anticipated to become a useful supplementary marker in clinical diagnostic settings. These clinical studies have also discovered new challenges relating to differences in analytical methods and the need for technical standardization, limitations in sample size and heterogeneous diseases, difficulties in adjusting urinary pteridines to urine concentration-dilution and adjusting for the natural variation of pteridine derivatives in urine. We anticipate in the next several years new translational studies to address these emerging clinical challenges as well as new research on the biological functions of peripheral pteridine derivatives. In this way, the study of urinary pteridines as disease biomarkers exemplifies broader efforts in the emerging field of urinary metabolomics as a new biomarker discovery platform.
Acknowledgments
This study was supported by a National Science Foundation Graduate Research Fellowship (#DGE-1011744), Department of Chemistry and Center for Single Nanoparticle, Single Cell, and Single Molecule Monitoring at Missouri University of Science and Technology.
Conflict of interest statement: All authors have declared no conflicts of interest. All authors contributed to the manuscript and approved its final version.
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