WO2008002911A2

WO2008002911A2 - Urate metabolites as diagnostic markers for cardiovascular and renal disease

Info

Publication number: WO2008002911A2
Application number: PCT/US2007/072116
Authority: WO
Inventors: Richard J. Johnson; George Henderson; Alexander Angerhofer
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2006-06-26
Filing date: 2007-06-26
Publication date: 2008-01-03
Also published as: WO2008002911A3

Abstract

Disclosed herein are methods of detecting and/or measuring uric acid metabolites in a biological sample. Also disclosed herein are methods of predicting risk of disease, monitoring progression of disease and diagnosing disease by detecting and/or measuring one or more uric acid metabolites in a biological sample. In addition treatment by antioxidants is being disclosed.

Description

URATE METABOLITES AS DIAGNOSTIC MARKERS FOR CARDIOVASCULAR AND

RENAL DISEASE

Related Applications

This application claims the benefit of U. S Serial No. 60/805,802 filed June 26, 2006, which is incorporated herein in its entirety.

BACKGROUND

[01] There is much debate about whether the presence of Uric Acid (UA) in the body is beneficial, neutral or detrimental to one's health. On the one hand, UA is believed to be an important antioxidant in the body (1 , 11-14). However, on the other hand recent research indicates that uncontrolled chronic hyperuricemia can lead to a number of health problems, including, but not limited to, hypertension, preeclampsia, kidney disease, metabolic syndrome, cardiovascular disease and diabetes. (2-10) It is the inventors belief that, in view of their recent research, and ongoing studies, that UA's causative effect for deleterious health consequences will ultimately become widely accepted in the medical community. Based on this, in depth characterization of the synthesis and especially metabolism of UA may lead to the elucidation of important biochemical markers that would allow the prediction of risk for disease, monitoring disease progression, and/or diagnosis of diseases for which UA is being shown to be a causative factor. Furthermore, such information may elucidate potential targets for treatments or prevention of disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[02] FIG. 1 Radical adduct formation in the reaction of peroxynitrite with uric acid

Fig. 2A: Systolic BP correlates with increased uric acid levels in rats. Rats were fed 2% oxonic acid, oxonic acid will allopurinol, oxonic acid with benziodarone or oxonic acid on low salt diet.

[03] Fig. 2B: SBP correlates with serum uric acid in children. Shown is the correlation between uric acid and systolic BP in 63 children. The correlation could not be explained by differences in body mass index or renal function

[04] Fig. 3: Formation of two carbon- based radicals in BPN spin trapping of the reaction of peroxynitrite with uric acid

[05] Fig. 4: LC-MS analysis (X axis= retention time; Y axis = relative abundance) of time course of the reactions of UA with PN and formation and disappearance of two reactive intermediates to give triuret. A=uric acid; B= M+1 215.9 (216); C=triuret; D= M+1 230.9 (231 )

[06] Fig. 5: Reaction scheme of the reaction of peroxynitrite with uric acid and in aqueous buffer or in MeOH/H₂O

[07] Fig. 6: LC-MS analysis (total ion current) of the reaction of uric in the presence of 1 equivalent of ASC and 1 or 2 equivalent peroxynitrite

[08] Fig. 7: Percent of uric acid remaining after the reaction with nitric oxide in the presence of various antioxidants

[09] Fig. 8: LC-MS identification of ¹⁵N-labeled triuret from plasma reactions of ¹⁵N- labeled uric acid with peroxynitrite. Panel A shows the total ion chromatogram of the plasma extract, Panel B shows plot of m/z149 and Panel C shows the ions of triuret peak at 4.76 min (Panel A). M/z 149 is the M+1 ion of labeled triuret and m/z 166 is the ammonium adduct of labeled triuret.

[010] Fig. 9: LC-MS/MS identification of ¹⁵N-labeled 6-aminouracil (6-AU) (Ret. Time = 5.55 min) from plasma reactions of ¹⁵N-UA with NO. Top two panels show the SRM for unlabeled 6-AU (formed from endogenous UA) and the bottom three show those for labeled (labeled has one extra SRM) 6-AU.

[011] Fig. 10: Mean plasma uric acid kinetics in rats using labeled uric acid

[012] Fig. 1 1 : Urine concentrations of uric acid and its metabolites in normal pregnancy (N=7) and in Pre-Eclampsia (N=9)

[013] Fig. 12: Pathways of uric acid reaction with peroxynitrite and oxidant

[014] FIG. 13 represents a table showing the results of plasma analysis of plasma dialysis patients and normal patients.

[015] FIG. 14 shows the LC-MS analysis of dialysis patient 1.

[016] FIG. 15 shows the LC-MS analysis of dialysis patient 2.

[017] FIG. 16 shows the LC-MS analysis of normal patient 1.

[018] FIG. 18 shows the LC-MS analysis of normal patient 2.

DETAILED DESCRIPTION

[019] The inventors have elucidated a number of physiologically relevant, biochemical pathways involving UA as a reagent. It has been found that UA can react with a number of oxidants produced in the body, resulting in the production of certain UA metabolite products. Based on such studies, the type of oxidants being produced in a subject can be elucidated by detecting and/or measuring UA metabolites in biological samples obtained from an individual. Furthermore, based on related studies showing that UA disrupts endothelial function and/or decreases nitric oxide (NO) levels, and in turn mediates detrimental health consequences discussed above, detecting and/or measuring UA metabolites enables determining risks of disease, progression of disease and disease status.

[020] According to one embodiment, the subject invention comprises detecting or measuring one or more uric acid metabolites in a biological sample and utilizing such information to determine a risk for developing a disease condition, to monitor progression of a disease condition, or to diagnose a disease condition. Biological samples used may be any suitable sample, including, urine, serum with or without other blood components, or tissue samples. A qualitative test can distinguish between the presence or absence of one or more UA metabolites, or can distinguish between categories of UA metabolite levels in a sample, such as absent, low concentration, medium concentration or high concentration. A quantitative test can provide a numerical measure of a UA metabolite in a sample.

[021] Another embodiment of the invention pertains to a method of determining an increased risk of metabolic syndrome in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein if said at least one uric acid metabolite is greater than a predetermined concentration is indicative of an increased risk of developing metabolic syndrome.

[022] A further embodiment pertains to a method of detecting an increased risk of cardiovascular disease in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein if said at least one uric acid metabolite is greater than a predetermined concentration is indicative of an increased risk of cardiovascular disease.

[023] A method of assisting diagnosis of or monitoring of a disease state in a subject, including hypertension, cardiovascular disease, preeclampsia, kidney disease, obesity, insulin resistance, diabetes or metabolic syndrome, related to hyperuricemia, the method comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein if said at least one uric acid metabolite is greater than a predetermined concentration is indicative of disease.

[024] In view of Applicants discovery that certain uric acid metabolites are present in biological samples and that such metabolites are indicative of reactions of uric acid with certain oxidants, the determination of elevated concentrations, those skilled in the art will be able to determine ranges and concentrations of a particular metabolite indicative or predictive for a certain medical condition.

[025] Described herein are techniques for detecting and/or measuring UA metabolites. In view of the teachings herein, those skilled in the art will appreciate that other techniques known in the art for detecting and measuring analytes can be configured for detecting UA metabolites. Such techniques include, but are not limited to, immunochromatographic techniques, HPLC, LC-MS, GC-MS and electrochemical detectors. In a specific embodiment, the disposable reagent unit comprises an assay chip. For example, the assay chip as taught in "Integration of Chemical and Biochemical Analysis Systems into a Glass Microchip" Analytical Sciences, January 2003 Vol. 19 (whose teachings are incorporated by reference) is adapted and configured for removeably engaging a diagnostic apparatus. The diagnostic apparatus comprises a light source that is directed to the developed portion of the immunoassay chip to produce a reflectance, or fluorescence, colorimetric or other signal that is detected by a sensor integrated into the diagnostic apparatus and processed to provide a value of the UA metabolite amount and/or presence. Further, those skilled in the art, in view of the teachings herein, will appreciate that the disposable reagent unit can take the form of a simple test strip similar to that used in conventional self-monitoring systems. For example, the test strip device as disclosed in U.S. Patent No. 6,352,862 is adapted to produce a signal corresponding to presence of the UA metabolite.

[026] The inventors' discovery also allows for the determination of whether ratios among UA metabolites change as a result of certain medical and physiological conditions. For example, the amount of 6-aminouracil, triuret or allantoin, when compared to one of the other may change as a result of a certain oxidative stress condition, cardiovascular disease, hypertension, diabetes, etc. Not only can metabolite/metabolite ratios be obtained but UA metabolite amounts can be compared to other biological indicators such as, but not limited to, serum or urine potassium, serum or urine glucose , cholesterol, triglycerides, blood urea nitrogen (BUN), sodium, chloride, bicarbonate (HCO₃ ^") and magnesium (Mg⁺⁺); serum and urine creatinine (Cr), uric acid and urine protein concentration. The invention enables the observance of such ratios as associated with a given medical condition. Additionally, the affect on any of the foregoing by elevated levels of UA metabolites can be tested.

[027] Experimental hyperuricemia (induced by inhibition of uricase with oxonic acid) results in the development of hypertension, salt-sensitivity, intrarenal vascular disease, and chronic renal injury (3-5). Fructose-induced metabolic syndrome is mediated largely by UA (8,9). In human studies, it has been demonstrated that 90% of new onset hypertension in adolescents is associated with elevated UA and in pilot studies the inventors have normalized blood pressure in 4 of 5 adolescents by lowering UA levels with allopurinol (6, 7). A comparison of UA levels with systolic blood pressure in the rat (Fig. 2A, (3)) and in new onset hypertension in humans (Fig. 2B, (6)) is presented to reveal the remarkable similarity.

[028] Evidence that it is the UA that is mediating these effects is provided by the finding that lowering UA either with a uricosuric (e.g., benziodarone) or a xanthine oxidase inhibitor (allopurinol) have equivalent effects on preventing hypertension, metabolic syndrome or renal disease (3, 5, 8). This is relevant since xanthine oxidase will generate both UA and O₂ ^", and hence agents such as allopurinol were historically used to block oxidants generated by xanthine oxidase. However, this supposition with allopurinol is likely incorrect. Allopurinol is actually a purine derivative that competes with xanthine for the reaction with xanthine oxidase. As such, its reaction will generate alloxanthine instead of UA; however, studies have demonstrated that it still generates oxidants in the process (15).

[029] UA can react with a variety of oxidants, including SO anion (O₂ ^"), hydrogen peroxide (H₂O₂), hydroxyl radical ('OH), and PN (0ONO^"). The reaction with the greatest affinity was thought to involve the reaction of UA with PN (16, 17). These reactions are initiated by the donation of an electron by UA to generate the urate radical (with a redox potential of 0.59V) followed by its nonreversible degradation to a variety of products (17, 21 ). In this regard the urate reaction is distinct from ascorbate, for although ascorbate will also generate the ascorbyl radical, this latter reaction is reversible.

[030] The antioxidant properties of UA can easily be demonstrated using in vitro culture systems. For example, PN is generated by the diffusion and rate-limited reaction of O₂ ^" and NO. The latter reaction frequently occurs in the vascular department due to the presence of O₂ ^" (generated by NADPH oxidase or xanthine oxidase) and NO (generated by endothelial NO synthase (eNOS)). PN may cause oxidative damage, including the nitrosylation of tissues (nitrotyrosine formation), that may have a role in the development of vascular disease. Incubation of endothelial cells with PN rapidly causes oxidative stress, leading to oxidation of tetrahydrobioptehn (BH4), which is critical in the enzymatic reaction by which eNOS generates NO. As a consequence, eNOS becomes 'uncoupled' and generates SO instead. The addition of UA with the PN can prevent the oxidative injury and partially restore the NO generation by eNOS, and complete restoration occurs when both ascorbate and UA are added (16). Additionally, UA may help maintain extracellular levels of SO dismutase by preventing its degradation by H₂O₂; this is another antioxidant thought to be important in protecting the extracellular milieu (20). Thus, these studies suggest that UA is good, and has led to the conclusion that elevated UA levels in CV disease may represent a beneficial host response to the oxidative stress known to occur in these conditions (11 , 12). However, as discussed above, these findings are marred by experimental data in animals showing that UA can cause hypertension and metabolic syndrome (see above). While UA may serve as an antioxidant, its reaction with PN or H₂O₂-based systems also generates carbon-based radicals that can be captured (Fig. 1) using spin-traps (18, 19).

[031] Santos et al. observed a radical by ESR spin-trapping with the spin trap DMPO in reaction mixtures of UA and PN (18). It was identified as the aminocarbonyl radical and its presence explained due to follow-up reactions between PN and the primary reaction products such as allantoin (18). The inventors' studies document more than one carbon- based radical species in this reaction when using PBN as the spin trap. Based on the hyperfine coupling constants and preliminary MS data, one of these trapped radicals is the aminocarbonyl radical. The fact that urate can be oxidized via a radical mechanism is significant since this opens up the possibility for an explanation of the pro-oxidant effect of UA. If the oxidation of UA in vivo produces carbon-based organic radicals, it is possible that radical chain reactions may be started that contribute to the various disease processes in which hyperuricemia is implicated. The conditions of the surrounding milieu, particularly the presence of chain-terminating antioxidants may, in fact, ameliorate these harmful chemical reactions under normal conditions and thus help to sustain the anti-oxidant properties of serum UA. In the absence of other antioxidants and/or when serum UA concentrations are elevated this protective effect may weaken and expose the negative side of UA. In support of this, the observation that ascorbate or NO (which can act as radical chain-breakers) can block the pro-oxidant effect of UA on Cu-mediated LDL oxidation. The protective effect of ascorbate on urate oxidation reactions may also occur in vivo. Naidoo and Lux observed a reduction in serum allantoin levels after oral administration of ascorbate, which indicates that UA oxidation was reduced (22). The possibility that ascorbate may be able to prevent some of the pro-oxidant effects of UA is also supported by the fact that the redox potential of the urate/urate radical pair is 0.59 V (23), which is higher than the corresponding redox potential of the ascorbate/dehydroascorbate pair (24,25).

[032] Without being held to any particular theory, the inventors believe that the mechanism(s) by which UA can cause CV disease is initiated by reactions of UA with various oxidants to generate radicals and other reactive intermediates or by the direct reaction of UA with nitric oxide itself (see below). In this regard, the inventors present data showing the following:

• When urate is incubated directly with endothelial cells, it has the effect to lower NO levels • Using ESR with two different spin traps (PBN and DMPO), several carbon-based radicals are generated during reaction of UA with PN. The inventors have also characterized the pathway of degradation using mass spectrometry (MS).

• Data has been generated showing that UA also reacts directly with NO, resulting in the degradation of NO and the generation of the unique product, 6-aminouracil (6- AU). This reaction occurs with greater affinity than that with PN or H₂O₂. It is further demonstrated that 6-AU is generated from this reaction in human plasma and is present in vivo in animals and humans.

• Data has been generated that show the formation of triuret in from PN reactions with UA, in human plasma, in rats and in human biological samples. This represents unique product of UA's reaction with PN and a biomarker for PN inducted oxidative stress.

• Antioxidants ascorbate and glutathione are shown to block specific pathway UA's reaction with oxidants (PN and NO reactions, respectively). This opens up the possibility of treatment with judiciously selected antioxidant cocktail that includes ascorbate and glutathione precursors.

[033] Thus, it is believed that UA may function like a "Trojan Horse", in that its reactions outside the cell may be relatively innocuous. However, once inside the cell the UA may both inactivate NO and generate radicals and intermediates with oxidizing and alklyating potential, leading to intracellular activation and a proinflammatory phenotype that causes a cascade of events culminating in CV disease.

Example 1: UA Reaction with PN: Generation of Carbon-Based Radicals

[034] Using Electron Spin Resonance (ESR), at least two different carbon-based radicals were detected when PN was reacted with UA in the presence of spin traps (Fig.3). The spin trap of choice was PBN (N-tert-butyl-α-phenylnitrone) since it gave no detectable background spectrum and yielded sufficiently strong ESR signals to allow for very low modulation amplitudes and correspondingly high spectral resolution. The hyperfine coupling constants are consistent with the aminocarbonyl radical which was observed previously using DMPO as the spin trap (18). Control experiments with the typical urate oxidation products allantoin, triuret, alloxan, and parabanic acid (16,27) have not yielded any ESR- detectable radical with the PBN trap under the same conditions (data not shown) indicating that these radical are formed from UA. Example 2: 5 UA Reaction Products with PN: Studies Based on Mass Spectrometry (MS)

[035] A series of reactions of UA with PN were conducted with both unlabeled and labeled ¹⁵N-UA and analyzed by LC-MS. The parallel use of labeled and unlabeled UA permitted the identification of UA derived products and key intermediates by LC-MS and LC- MS/MS. In all experiments PN was shielded from light and kept on ice. UA was added to a K phosphate buffer typically at pH 7.4 (some the reactions were conducted at higher pH and the pH had no effect on the products suggesting that the reaction occurs with the mono anion of UA). Typically one equivalent of PN was then added to the reaction mixture. The reaction mixture was vortexed for 10 sec, allowed to incubate at room temperature for 3 min, placed on ice and transferred to LC-MS vials. The vials were immediately frozen at -8O⁰C until LC-MS analysis. The final concentrations of UA were 300μM (physiological) to 10 mM and PN concentrations were either 1 , or 3 equivalents to UA. In the concentration range investigated the UA concentration had no effect on the products formed. Treatment with one equivalent of PN results in complete reaction of UA.

[036] The LC-MS analyses were performed using Finnigan triple quadrupole mass spectrometer model TSQ 7000 and Agilent quaternary pump HPLC model 1100, using Phenominex Luna C18 column (either 150 or 250x4.6 mm). Samples were kept cold until analysis to minimize the decomposition of labile intermediates. The mobile phases used in the LC separation of UA reaction products included NH₄OAc/AcOH and methanol (as gradient), formic acid/Methanol gradient or NH₄OAc/AcOH /acetonitrile gradient. Evaluation of the molecular weight and fragmentation patterns (which yield structural information about the molecules) of the intermediates was performed using the mass spectrometer after the compounds had been separated from one another on the LC column. The ionization method used in performing the mass spectroscopy was +ive and -ive APCI (atmospheric chemical ionization) ionization. This ensured that we were able to clearly visualize the molecular weight and fragmentation pattern of each compound, as some compounds are more easily ionized (and hence more easily detected) to positive ions, and some are more easily ionized into negative ions. Mass spectrometric scans were performed in the fullscan, ms-ms (tandem) and SRM modes. This enabled us to detect all ions present as well as the unique fragmentation pattern of each intermediate/product present. In order to determine the exact quantities of each compound present in the reaction mixture, the SRM mode was used. SRM is one of the best ways to perform LC-MS quantitation. SRM provides a unique fragment ion that can be monitored and quantified in the midst of a very complicated matrix. This characteristic makes the SRM plot ideal for sensitive and specific quantitation. [037] Fig. 4 shows the full scan LC-MS total ion trace of one such reaction which initially produced two unstable intermediates with a M+1 =216 (M+1 refers to the mass of the protonated, or positively charged intermediate formed during the ionization process of the mass spectrometry) (product B; RT=4.93 min) and 231 (product D; RT=6.01 min). By 1hr at room temperature most of the products B and D had disappeared. After 16 hrs at room temperature, all the starting materials and intermediates were converted to triuret (C). From several studies conducted in aqueous medium, it is evident that the formation of triuret is a signature reaction of PN with UA in aqueous medium. Subsequent analysis (MS/MS and different solvents) showed that product B was the primary intermediate and the product D was partially formed in the HPLC when MeOH was used as the mobile phase. The reaction scheme is shown in Fig 5. In the presence of MeOH [either added before or after the reaction] the formation of both products and subsequent formation of alkylation product E with M+1 of 173 was identified by MS (from products of labeled UA and methylated homologs) to have the structure shown in Fig. 5. When UA labeled with ¹⁵N at positions 1 and 3 was used, all the products observed, including triuret were doubly labeled suggesting that they all came from the six membered ring of UA. Additional reactions were conducted with Li urate at physiological pH (similar to IV solutions used later in the study) and they gave the same products as in phosphate buffered reactions.

[038] UA-PN Reactions in the Presence of Antioxidants: We examined the reactions of UA and Li urate with PN in phosphate buffer in the presence of selected biologically important antioxidants including ascorbic acid (ASC), tetrahydrobiopterin (BH₄), glutathione (GSH), cysteine (CYS) and tyrosine (TYR). In reactions conducted with 1 :1 :1 ratio of antioxidants, UA and PN, the following percent of UA remained (UA remaining is a measure of protection of UA by a given antioxidant): ASC (101 %), BH₄ (8%), GSH(37%), CYS(21%) and TYR(I %). Fig. 6 shows the reaction of UA in the presence of 1 equivalent of ASC and 1 or 2 equivalents of PN. Thus, among the antioxidants tested, only ASC affords the complete protection of UA from an equal amount of PN.

Example 3: UA Reaction with NO

[039] In a typical reaction, UA solution was combined with K phosphate buffer to prepare solutions for reaction with NO gas (Air Liquide). Final reaction volumes were 3mL for each reaction and the final concentrations of UA varied from 300 μM (physiological) to 10 mM. The reaction mixtures were purged of oxygen by bubbling with argon (3 min), and were then placed on ice, protected from light. The mixtures were bubbled with NO gas for 10, 30 or 90 sec. The reaction mixtures were then sealed and kept at room temperature for a total of 3 min. The mixtures were then placed on ice, transferred into MS vials and vials were then frozen at -8O⁰C until LC-MS analysis. LC-MS analyses used either NH₄Ac/AcOH or formic acid and MeOH as mobile phases. These studies show that UA was rapidly converted to a single compound with M+1 ion of 128 which was subsequently identified as 6-AU. Some reactions were conducted using Li urate at pH 7.4 and they also produced only 6-AU. This reaction to produce 6-AU occurs even in the presence of PN or H₂O₂. The reaction product in reaction mixture was stable for at least a month at ambient temperature. When the reaction (short NO bubbling) mixtures that contained unreacted UA were allowed to stand at ambient temperature, no further reaction occurred, suggesting that NO (no additional NO becomes available after the initial reaction) was consumed in the reaction (if it is not consumed further reaction will occur). When the UA was labeled with ¹⁵N at positions 1 and 3, all the 6-AU produced was doubly labeled, suggesting that it came from the six membered ring of UA. This rapid reaction of NO with UA provides a novel mechanism for the depletion of NO in the biological system and the fact that this reaction occurs exclusively even in the presence of PN or H₂O₂ indicates that NO depletion can occur in the presence of high levels of UA even in the absence of significant amount of PN (previously it was thought that NO acted on UA through the formation of PN). This also provides a unique opportunity to monitor the direct reaction pathway of NO with UA by measuring the amount of 6-AU produced in vivo.

[040] UA-NO Reactions in the Presence of Antioxidants: We have investigated the reactions of UA and Li urate with NO in the presence of selected antioxidants ASC, BH₄, GSH, CYS and TYR. These reactions were conducted by bubbling NO for various time periods (10, 30, 90 sec). Investigations conducted with UA in buffered solutions and NO showed that 30 sec bubbling provides 1 equivalent of NO and it resulted in an essentially complete reaction of UA (10 mM UA, 3 ml solution). Further bubbling had no effect on the product formed when UA was used alone. We have conducted comparative antioxidant studies with a 1 :1 :1 ratio of UA, antioxidant and NO. Fig. 7 shows the percent of UA remaining in reactions conducted in the presence of antioxidants. Among the antioxidants tested, one equivalent of GSH affords the best protection (76%) of UA from NO reaction. Thus, it is possible to block the UA-NO reaction by GSH, at least partially. Example 4: UA Reaction with SO

[041] The reaction of UA with SO and SO generating systems have been reported to yield ailantoin as the primary or sole product (27). We have conducted limited studies with UA in phosphate buffer and potassium SO dissolved in DMSO (KO₂ reacts with water). These reactions produced ailantoin as the only identifiable product (~ 90%) in LC-MS analysis (identified by its retention time and characteristic mass spectrum in LC-MS). Example 5: Reactions of Stable Isotope Labeled UA with PN and NO in Human Plasma

[042] Plasma PN Reaction: ¹⁵N-UA added to human plasma from healthy donors was incubated with PN at 1 to 6 times the equivalent of labeled UA. At the end of reaction (3 min), the plasma samples were deproteinized with 3X volume of 20% TCA and filtered through 0.2μ filter and the filtrate was analyzed by LC-MS in the full scan mode and SRM. Labeled triuret from UA was the only identifiable product. No significant amounts of other labeled products including allantoin or 6-AU were present in the plasma reaction. If there were any products of alkylation of plasma proteins, they would not be identified by the method employed (proteins are removed before analysis). Fig. 8 shows the identification of labeled triuret from plasma reactions of labeled UA with PN. The exclusive formation of triuret from the reaction of UA from PN, even in the presence of plasma components suggests that measuring triuret concentration can indicate the extent of PN reaction in vitro and in vivo. Similar results were obtained when the reactions were performed with cell lysates.

[043] NO Reaction: Labeled UA spiked plasma reactions were performed with NO gas bubbled for different periods starting from 30 sec to 6 min. The plasma samples were deproteinized (as before) and analyzed by LC-MS in the full scan and SRM modes. In all experiments UA produced only labeled 6-AU. No other UA products were identified. Fig. 9 shows the identification of labeled 6-AU from plasma reactions of ¹⁵N-UA with NO (multiple ions specific for labeled and unlabeled (endogenous) 6-AU were monitored, thus ensuring accurate identification). The formation of 6-AU provides a means of determining the extent of direct reaction of NO with UA.

Example 6: LC-MS/MS Method for the Analysis of UA, Allantoin, Triuret, Alloxan, and Parabanic Acid in Biological Samples.

[044] The inventors have developed LC-MS/MS methods that can identify and quantify key UA products in biological samples. This method uses TSQ 7000 LC-MS in the APCI (atmospheric pressure chemical ionization) positive (allantoin, triuret and 6-AU) and negative mode (UA₁ alloxan and parabanic acid). This method is crucial for the conduct of the proposed studies. In reality the LC-MS method has to be run twice (once in the positive mode and once in the negative mode) to measure all the compounds of interest. Typical method uses a 150x4.6 mm Phenomenex 5 μ Luna column at a flow rate of 0.6ml per min. The mobile phase consists of 5mM NH₄Ac in 0.1% acetic acid (solvent A) and MeOH (solvent). A 15 or 10 min gradient was used for most analyses (starting from 95% A and 5% B to 70%A and 30%B). For general LC-MS analyses gradients up to 95%B were used. Multiple characteristic SRMs for each of the labeled and unlabeled compounds were used for identification and quantitation of various UA products. The method has been developed for the analyses of both plasma (deproteinized with 3X volume of TCA and filtered) and urine samples (diluted 3X and filtered). For the determination of low levels of UA in cell culture experiments, we adapted this method for use with negative electrospray ionization (ESI) mode. The method can readily measure UA up to 1nM. Further tests have shown that even lower levels of allantoin, triuret and 6-AU can be measured in the positive ESI mode. None of the studies thus far had indicated the formation of any significant amounts of parabanic acid or alloxan and these compounds will not be further studied in our project.

Example 7: Studies with Labeled UA in Rats

[045] Pilot rat studies were conducted using four male Sprague Dawley rats to determine the kinetics and metabolism (UA breakdown and metabolism products) of labeled ¹⁵N-UA. The IV solution of UA was prepared using LiOH and glucose and the final isotonic solution contained 6mg/m! of ¹⁵N-UA, 37.83 mg/ml of glucose, and 0.22 mg/ml of free Li (0.025%). It was sterile filtered before use. Rats underwent jugular vein cannulation the day before blood samples were taken to study UA kinetics. The cannula remained in place for two and a half days. On the study day, 0 time urine (from previous 24 hr) and plasma (0.3 ml blood from cannula) were collected. The study was started by tail vein injection (1ml/200g rat) of ¹⁵N-UA solution. Plasma samples were collected at 15min, 1 hr, 4 hr, 8hr, 24hr after IV injection and urine samples were collected from 0-8 hrs and 8-16 hrs. The plasma samples (100 μl) were deproteinized with TCA solution and filtered through 0.2μ filter and analyzed by LC-MS as described in Section C.10. The urine samples were filtered through 0.2μ filter and analyzed by LC-MS. Fig. 10 shows the kinetics data obtained using the ¹⁵N-UA. The data were subjected to pharmacokinetic modeling using Winnonlin software. The data fitted a one compartment model and UA had mean plasma half life of 6.1 min. In the mass spectral analysis of the plasma and urine from rats treated with labeled UA, labeled 6-AU, allantoin and triuret were identified as metabolites. No labeled parabanic acid or alloxan were identified in these pilot studies.

Example 8: Analysis of Urine Samples from Volunteers with Normal Pregnancy and from Patients with Pre-Eclampsia.

[046] Pre-eclampsia is a disorder characterized by pregnancy-induced hypertension and both increased oxidative stress and reduced antioxidant defenses. The LC-MS/MS analysis method (see Section C.10) developed as part of the preliminary studies for this project was applied for the analysis of representative urine samples (samples were diluted 3X and filtered through 0.2μm filter) from pre-eclampsia patients and normal pregnancy controls. The levels of various metabolites were identified and quantified using calibration standards of the metabolites. The mean values and standard deviations are presented in Fig. 11. The significant findings are that there is a substantial amount of triuret (to the inventors' knowledge, no one has reported the presence of triuret in any human studies before) excreted in urine. In addition to allantoin, a significant increase in 6-AU (the newly identified product of the NO reaction with UA) was also present in the preeclamptic urine compared to controls. No significant amounts of parabanic acid or alloxan were excreted in either group.

[047] As discussed above, relative, qualitative measurements of a particular UA metabolite concentration relating to low risk vs. high risk patients for a disease state, or indicative of a disease state may be determined, in view of the teachings herein. Quantitative ranges may also be determined for prediction or indication of a disease state, in view of the teachings herein. Based on preliminary studies, and not to be bound by specific concentrations, increased concentrations of aminouracil in urine may pertain to concentrations higher than 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5,

1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6,

3.7, 3.8, 3.9, or 4.0 mg/dl; increased concentrations of triuret in urine may pertain to concentrations higher than 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0 mg/dl; increased concentrations of allantoin may pertain to concentrations higher than 9.0, 9.5, 10.0, 10.5, 1 1.0, 11.5, 12.0, 12.5, 13.0, 13.5 or 14.0 mg/dl. Not to be bound by any specific range, preliminary data in normal pregnant patients vs. preeclampsia patients suggests that normal concentrations in pregnant women fall in the range of 0.6-1.1 mg/dl in the urine, and >1.2 mg/dl in urine preeclampsia patients for aminouracil; for triuret, 5-7 mg/dl in normal pregnancy and >8 in preeclampsia; for allantoin, concentration may be as high as 8-12 mg/dl in normal pregnancy and 10-20 mg/dl in preeclampsia.

Example 9: Summary and Integration of Preliminary Studies. [048] Without being held to any particular theory, the in vitro studies discussed herein have shown three key pathways (Fig. 12) for the biological oxidation of urate, all associated with signature end-products. Data indicates that these 3 pathways occur in animals and humans, with evidence for activation of the PN-UA and NO-UA reactions in subjects with preeclampsia. The latter studies also indicate that these degradation pathways may account for much of the disposal of uric acid, as the sum concentration of these products was similar to the concentration of uric acid present in the urine (Fig 15). The action of uricase enzyme or SO predominantly oxidizes UA to allantoin whereas the reaction with PN produces reactive intermediates that are converted to triuret in aqueous medium. In the presence of alcohol (and potentially biomolecules containing removable hydrogen such as compounds containing -OH, -SH and -NH₂ groups) the urate-PN reaction resulted in the alkylation of such molecules by urate intermediates. Measurement of allantoin and triuret can be utilized to differentiate the pathways of oxidation of UA in vivo This provides identifiable pathways for the potential deleterious effects of excess UA in vivo. Triuret can also be an ideal biomarker for the reaction of UA with PN.

[049] The inventors' have led to the discovery that NO reacts with UA to produce 6- AU and this unique reaction occurs even in the presence of PN, H₂O₂, suggesting that a pathway for NO depletion exists independent of reactions with PN or SO. Since NO is an important regulator of vasculature, the identification of a selective reaction with UA offers an in vivo mechanism for the depletion of NO, especially in the presence of excess UA. While UA is always present in plasma, NO has a short half-life and may react with many substances. Therefore, the reaction of UA and NO may be limited to areas where both are present and in excess, as may occur inside the endothelial cell in the setting of hyperuricemia — particularly in the setting of oxidative stress in which intracellular levels of GSH are low.

[050] Based on the elucidation of the different metabolic pathways for uric acid, the treatments are devised intended for inhibiting one or more of such pathways. According to one embodiment, the subject invention pertains to a composition that inhibits metabolism of uric acid into aminouracil that comprises an effective amount of n-acetylcysteine, or some other glutathione generating compound and pharmaceutically acceptable carrier. In another embodiment, the subject invention pertains a composition containing a glutathione generating compound; vitamin C and a pharmaceutically acceptable carrier. The compositions may be formulated for oral administration or parenteral administration.

Example 10: Elevation of Uric Acid Metabolites in Dialysis Patients

[051] Plasma samples from two dialysis patients and two normal plasma patients was analyzed for the presence of 6-aminouracil, triuret, and allantoin. FIG. 13 represents a table showing the results of the plasma analysis. As shown, levels of 6-aminouracil and allantoin was substantially and significantly raised in the dialysis patients compared to normal patients. Particularly noteworthy is the finding that normal plasma had almost no detectable triuret compared to dialysis patients, which had exceptionally high levels of triuret. FIGs. 14- 17 represent graphs of the spectral analysis of dialysis patient plasma (FIGs 14 &15) and normal patients (FIGs. 16 & 17) using LC-MS. References

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[052] The disclosures of the cited patent documents, publications and references are incorporated herein in their entirety to the extent not inconsistent with the teachings herein. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of detecting uric acid metabolism in a subject comprising obtaining a biological sample from said subject; and testing said biological sample for an amount of at least one uric acid metabolite.

2. The method of claim 1 wherein said at least one uric acid metabolite is triuret, allantoin or 6-amino uracil

3. The method of claim 1 , wherein said at least one uric acid metabolite is 6-amino uracil.

4. The method of claim 1 wherein said biological sample comprises urine.

5. The method of claim 1 wherein said biological sample comprises serum or plasma.

6. The method of claim 1 wherein said at least one uric acid metabolite is triuret.

7. The method of claim 1 , wherein said at least one uric acid metabolite is allantoin.

8. A method of determining oxidative stress in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample.

9. The method of claim 8, wherein detecting triuret at or above a predetermined amount is indicative of peroxynitrite related oxidative stress.

10. The method of claim 8, wherein detecting allantoin at or above a predetermined amount is indicative of superoxide related oxidative stress.

11. A method of determining endothelial dysfunction due to uric acid mediated NO depletion in a subject comprising obtaining a biological sample from said subject; and testing for an amount of 6-amino uracil in said biological sample.

12. A method of determining an increased risk of metabolic syndrome in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein detecting at least one uric acid metabolite at or above a predetermined amount is indicative of an increased risk of developing metabolic syndrome.

13. The method of claim 12, wherein said biological sample is urine.

14. The method of claim 15, wherein said uric acid metabolite is 6-amino uracil.

15. A method of determining an increased risk of cardiovascular disease in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein detecting at least one uric acid metabolite at or above a predetermined amount is indicative of an increased risk of cardiovascular disease.

16. The method of claim 15, wherein said at least one uric acid metabolite is 6- aminouracil.

17. A method of determining biological effects of increased levels of a uric acid metabolite comprising administering an amount of said uric acid metabolite to a nonhuman animal according to a predetermined regimen; and observing changes in at least one biological parameter in said nonhuman animal; wherein said uric acid metabolite is 6-aminouracil, triuret, or allantoin, or a combination thereof.

18. The method of claim 17, wherein said predetermined regimen comprises at least one dose a day for at least one week.

19. The method of claim 17, wherein said at least one biological parameter comprises blood pressure, weight, serum or urine potassium, serum or urine glucose , cholesterol, triglycerides, blood urea nitrogen (BUN), sodium, chloride, bicarbonate (HCO₃ ^") and magnesium (Mg⁺⁺); serum and urine creatinine (Cr), uric acid and urine protein concentration.

20. A method of diagnosing or predicting preeclampsia comprising obtaining a biological sample from a patient; and testing said sample for at least one uric acid metabolite, wherein detecting a uric acid metabolite at or above a predetermined amount is indicative of preeclampsia or increased risk of preeclampsia.

21. The method of claim 20, wherein said at least one uric acid metabolite is triuret or 6-aminouracil, or both.

22. A method of testing antioxidant compounds for amelioration of peroxynitrite related oxidative stress comprising administering a test antioxidant compound to a subject undergoing peroxynitrite- related oxidative stress; and monitoring an effect of said test antioxidant compound on triuret levels in said subject.

23. A method of testing antioxidant compounds for amelioration of superoxide related oxidative stress comprising administering a test antioxidant compound to a subject undergoing superoxide- related oxidative stress; and monitoring an effect of said test antioxidant compound on allantoin levels in said subject.

24. A method of determining an increased risk of metabolic syndrome in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein detecting at least one uric acid metabolite at or above a predetermined amount is indicative of an increased risk of developing metabolic syndrome.

25. A method of detecting an increased risk of cardiovascular disease in a subject comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein detecting at least one uric acid metabolite at or above a predetermined concentration is indicative of an increased risk of cardiovascular disease.

26. A method monitoring of a disease state in a subject, said disease state being hypertension, cardiovascular disease, preeclampsia, kidney disease, obesity, insulin resistance, diabetes or metabolic syndrome, the method comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein if said at least one uric acid metabolite is at a predetermined concentration this is indicative of a correlative disease state.

27. A method of diagnosing a disease in a subject, said disease state being hypertension, cardiovascular disease, preeclampsia, kidney disease, obesity, insulin resistance, diabetes or metabolic syndrome, the method comprising obtaining a biological sample from said subject; and testing for an amount of at least one uric acid metabolite in said biological sample; wherein if said at least one uric acid metabolite is at a predetermined concentration this is indicative of a disease state.

28. A method of identifying a therapeutically-active compounds for amelioration of NO depletion comprising administering a test compound to a subject; and monitoring an effect of the test compound on a 6-aminouracil level in said subject, whereby the test compound is identified as therapeutically active if it causes a reduction in the 6-aminouracil level.