Purine metabolites

Purines contain two rings in their structure. The two purines commonly found in nucleic acids are adenine (A) and guanine (G) both are found in DNA and RNA. Other purine metabolites, not usually found in nucleic acids, indude xanthine, hypox-anthine, and uric add. 

Adenosine deaminase (ADA) was the first therapeutic enzyme coupled to PEG with the aim of reducing clearance and thereby overcoming the short half-life of ADA. Patients deficient in ADA are unable to regulate purine metabolism. As a result purine metabolites (e.g., adenosine monophosphate) accumulate to cytotoxic levels in B-lymphocytes and lead to severe B-cell depletion that presents clinically as severe combined immunodeficiency syndrome (SCIDS). While intramuscular injection of unmodified ADA provides some relief, antibodies develop rapidly against the protein and prevent it from being useful as replacement therapy. Even in the absence of antibodies, unmodified ADA s plasma half-life is only a few minutes. 

Xanthosine, which is derived from purine metabolites, is the first committed intermediate in caffeine biosynthesis (Fig. 4). Xanthosine can be formed from de novo purine biosynthesis, S-adenosylmethione (SAM) cofactor, the adenylate pool, and the guanylate pool (169). De novo purine biosynthesis and the... 

Adenosine, a natural purine metabolite of adenine nucleotides, is a key regulator of many physiologic functions including vascular blood flow, platelet thrombotic... 

Several papers can be found in the literature concerning the simultaneous determination of creatinine and uric acid or various purine metabolites however, only a few have reported the simultaneous determination of creatinine and purine derivatives. 

RP-HPLC procedures for the determination of creatinine and purine metabolites, such as allantoin, uric acid, xanthine, and hypoxanthine in ruminant urine, were described. Chromatography was achieved with a Cig column under isocratic conditions, and detection at 218 nm without allantoin derivatization. The chromatographic conditions were a compromise between the sensitivity and specificity of the measurement of each analyte, analysis time, and resolution of all analyte peaks from interfering compounds.Uremic toxins creatine, creatinine, uric acid, and xanthine were simultaneously determined in human biofluids, simply after dilution, with UV detection at 200 nm. This method was compared, for creatinine and uric acid, with conventional routine methods and did not give significantly different results. 

Purines are metabolized in a series of reactions involving hypoxanthine, xanthine, uric acid, and allantoin as end products that are subsequently excreted in urine. Fig. 3 shows the metabolic pathways for xanthine. Measurement of urinary excretion of purine metabolites, primarily allantoin or, additionally, uric acid, xanthine, and hypoxanthine, has been proposed as a marker for microbial... 

The combination of HPLC reversed-phase Cg columns with monitoring of the effluent at 205 nm provides an acceptable analytical tool for quantification of purine metabolites in the urine. Detection at short ultraviolet... 

UV) wavelengths up to 215 nm results in compromised method selectivity and, therefore, longer and more specific detection wavelengths were applied in recent methods. The combination of the direct separation and determination using a reversed-phase HPLC system, coupled with photodiode array detection, is found to be accurate and more precise and selective than previously published methods for the determination of purine metabolites in urine. 

In recent years, a number of methods based on HPLC, isocratic or gradient, have been reported for the quantification of allantoin and purine metabolites in biological fluids. 

Shingfield, K.J. Offer, N.W. Simultaneous determination of purine metabolites, creatinine and pseudouridine in mminant urine by reversed phase HPLC. J. Chromatogr., B 1999, 723, 81-94. 

Nucleotidase [15] followed by adenosine nucleosidase [16] are expected to be the enzymes responsible for the step-by-step conversion of the cytokinin nucleotide to the base iPA. Both of these reactions may proceed also in the opposite direction, and in this case they are catalysed by adenosine phosphorylase (ribosylation of iPA, [17]) and adenosine kinase (phosphorylation of iPAR, [18-20]). These enzymes are common in the mutual conversions of adenine and purine metabolites (reviewed in [21]) and their properties have been summarised by [22]. These enzyme activities seem to be the key for understanding the fate of C-labelled adenine (Ade) and adenosine (Ado) in feeding experiments [summarised by 23]. 

Reabsorption of purine metabolites occurs at high urinary pH An adult patient is being treated for acute leukemia with a combination of anticancer dmgs that includes cyclophosphamide, mercaptopurine, methotrexate, vincristine, and prednisone. He is also using dronabinol for emesis, a chlorhexidine mouthwash to reduce mucositis, and laxatives. The patient complains of pins and needles sensations in the extremities and muscle weakness. He is not able to execute a deep knee bend or get up out of a chair without using his arm muscles. He is also very constipated. If these problems are related to the chemotherapy, the most likely causative agent is (A) Cyclophosphamide Dronabinol Mercaptopurine Prednisone Vincristine... 

SCID patients, if untreated, usually die within 1 year due to severe, recurrent infections such as chronic diarrhea, ear infections, recurrent pneumonia, and profuse oral candidiasis. The ADA dehciency leads to an accumulation of purine metabolites, in particular dGTP, which are cytotoxic to lymphoid stem cells. Restoration of normal ADA levels removes the dGTP and allows the lymphoid stem cells to propagate. Pegademase is administered intravenously, stored at 4°C, and should never be frozen [77]. 

Fig. 3, Chromatographic profiles of purine metabolites extracted with acid from skin fibroblasts pulsed with C-hypoxanthine. 50 vl aliquot of extract from 7.4 x 10 cells of a normal control.

Several detection systems are utilized in CE for the analysis of nucleoside and nucleotide mixtures. The performances of UV-visible absorption, conductance, electrochemical, a- P radiochemical and fluorescence detectors and mass spectral interfacing have been compared recently. Although UV-visible absorption is generally considered as not very sensitive, low limits of detection (LODs) of 8x10 mol 1 have been reported for purine metabolites using this method. The conductivity technique suffers from poor sensitivity. Electrochemical detection has a higher sensitivity, but its usefulness is limited by the fact that only electroactive species can be detected. Detection by mass spectrometry (MS) leads to poor sensitivity and implies expensive instrumentation. Radiochemical detection has been applied to a- P-labeled thymidine, cytidine, and adenosine... 

Britz-McKibbin P, Nishioka T, and Terabe S (2003) Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis a new tool for metabolomic studies. Analytical Sciences 19 99-104. 

Uric acid and xanthines are markers for metabolic disorders such as gout, Lesch—Nyman syndrome, and xanthinuria. Measurements of urinary excretion of purine metabolites, among them uric acid and xanthine, have been proposed as a marker for microbial protein synthesis. Their simultaneous determination is useful for diagnosis and treatment of hyperuricemia. In addition to xanthine and hypoxanthine, notable members of the xanthine class include caffeine, theophylhne, and theobromine. ... 

Four purine metabolites (allantoin, uric acid, xanthine, and hypoxanthine) were isolated from ovine urine and analyzed on 2 x 250 x 4.6 mm Cjg columns (photodiode array detector, 2 = 225-284 nm). A complex 31-min 100/0 -> 95/5 water (2.5 mM ammonium phosphate to pH 3.5 with H3P04)/methanol gradient was used [1572]. Allantoin was poorly retained and minimally resolved from early-eluting coextracted components. The standard concentration range was 70-1500 pM with quantitation limits of 0.5-2 nmol injected (S/N = 10) reported. 

Purine metabolites in patient SB during different treatment periods. 

Table 1 Purine metabolites and enzyme levels in family B.

Becroft. Pyrimidine and purine metabolites in ornithine carbamoyltransferase deficiency. J.Inher.Metab.Dis. 4 27 (1981). 

PURINE METABOLITES, URACIL AND cAMP DURING EXCHANGE TRANSFUSION... 

Purine metabolites were measured via fluorimetric detection of the rate of H2O2 production during sequential catabolism of uric acid. H 02 oxidizes nonfluorescent dichlorofluorescin to fluorescent dichlorofluorescein under conditions that allow for detection of lOpmoles of purine substrate, Deoxy- and ribo-nucleosides may be partitioned by boronate affinity chromatography thus allowing their separate determination. 

D.R. Webster, H.A. Simmonds, D.M.J. Barry and D.M.O. Becroft. Pyrimidine and Purine Metabolites in Ornithine Carbamoyl Transferase Deficiency. 

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