Extended diagnosis of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical validation.

BACKGROUND AND AIMS: Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine. METHODS: The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders. RESULTS: Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens. CONCLUSIONS: A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.

Urinary concentrations of AICAR and mannitol in athlete population.

Both AICAR and mannitol are prohibited for use in sports, but no decisive criteria that would guide anti-doping laboratories on data interpretation have been established so far. In an attempt to help harmonize reporting and management of analytical findings, reference population data collected for US athletes are presented. Upon analysis of 12 377 samples, mean urinary AICAR concentration was found to be 647 ± 365 ng/mL with median value of 574 ng/mL, 99th percentile at 1786 ng/mL and 99.7th percentile at 2151 ng/mL. Based on these results, we suggest that any sample with AICAR concentration greater than 2000 or 2500 ng/mL be analyzed by carbon isotope ratio mass spectrometry to establish the origin. Urinary mannitol concentrations demonstrate larger variation with the mean value of 72 ± 140 µg/mL and median at 41 µg/mL (n = 6407). While the 99.7th percentile for mannitol was measured to be 1094 µg/mL, the population data alone is not sufficient to suggest a threshold value. It is also shown that the use of mannitol as a sweetener in amounts of up to 20 g per day results in a urinary concentration of about 14 mg/mL. As only intravenous mannitol is prohibited in sports, controlled excretion studies are needed to see whether intravenous administration could in fact be discriminated from dietary intake. An important observation is that mannitol present in mg/mL quantities significantly increases urine specific gravity, which makes a widely accepted normalization approach not applicable.

Urine levels of 5-aminoimidazole-4-carboxamide riboside (AICAR) in patients with type 2 diabetes.

AIMS: 5-Aminoimidazole-4-carboxamide riboside (AICAR) is an endogenous activator of AMPK, a central regulator of energy homeostasis. Loss and/or reduction of AMPK signaling plays an important role in the development of insulin resistance in type 2 diabetes. The loss of AMPK in diabetes could be due to a loss of AICAR. The aim of this study was to characterize urine levels of AICAR in diabetes and determine whether an association exists with respect to late complications, e.g., retinopathy, nephropathy and neuropathy. METHODS: Urine AICAR was measured by liquid chromatography tandem mass spectrometry in 223 patients consisting of 5 healthy controls, 63 patients with pre-diabetes, 29 patients with newly diagnosed type 2 diabetes and 126 patients with long-standing type 2 diabetes. For statistical analyses, nonparametric Kruskal-Wallis test, one-way ANOVA and multivariate regression analysis were performed to investigate the associations of urinary AICAR excretion within different groups and different clinical parameters. RESULTS: The mean urine AICAR for all 223 patients was 694.7 ± 641.1 ng/ml. There was no significant difference in urine AICAR between the control and patients with diabetes (592.3 ± 345.1 vs. 697.1 ± 646.5 ng/ml). No association between any of the biochemical and/or clinical parameters measured and urine AICAR was found, with the exception of age of patient (R = - 0.34; p < 0.01) and estimated glomerular filtration rate (R = 0.19; p = 0.039). These results were confirmed additionally by linear regression analysis. CONCLUSIONS: Clinical diabetes is not associated with a change in endogenous AICAR levels. Loss of AICAR may therefore not be a mechanism by which AMPK signaling is reduced in diabetes.

Doping control analysis of 46 polar drugs in horse plasma and urine using a 'dilute-and-shoot' ultra high performance liquid chromatography-high resolution mass spectrometry approach.

The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening method to detect 46 polar drugs in equine urine and plasma, including some angiotensin-converting enzyme (ACE) inhibitors, sympathomimetics, anti-epileptics, hemostatics, the new doping agent 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), as well as two threshold substances, namely dimethyl sulfoxide and theobromine. For plasma, the sample (200µL) was protein precipitated using trichloroacetic acid, and the resulting supernatant was diluted using Buffer A with an overall dilution factor of 3. For urine, the sample (20µL) was simply diluted 50-fold with Buffer A. The diluted plasma or urine sample was then analysed using a UHPLC-HRMS system in full-scan ESI mode. The assay was validated for qualitative identification purpose. This straightforward and reliable approach carried out in combination with other screening procedures has increased the efficiency of doping control analysis in the laboratory. Moreover, since the UHPLC-HRMS data were acquired in full-scan mode, the method could theoretically accommodate an unlimited number of existing and new doping agents, and would allow a retrospectively search for drugs that have not been targeted at the time of analysis.

"Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 µg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

Determination of ¹³C/¹² C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide 1ß-D-ribofuranoside (AICAR).

RATIONALE: AICAR (5-aminoimidazole-4-carboxamide 1ß-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated. METHODS: Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study. RESULTS: The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h. CONCLUSIONS: Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR.

Quantification of urinary AICAR concentrations as a matter of doping controls.

Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised (13)C(5)-labelled AICAR as internal standard.

In vivo proton MR spectroscopy findings specific for adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

Misleading behavioural phenotype with adenylosuccinate lyase deficiency.

Adenylosuccinate lyase deficiency is a rare autosomal disorder of de novo purine synthesis, which results in the accumulation of succinylpurines in body fluids. Patients with adenylosuccinate lyase deficiency show a variable combination of mental retardation, epilepsy and autistic features and are usually discovered during screens for unexplained encephalopathy using the Bratton-Marshall assay that reveals the excretion of the succinylaminoimidazolecarboxamide riboside (SAICAr). Here, we report on two sisters aged 11 and 12 years presented with global developmental delay, motor apraxia, severe speech deficits, seizures and behavioural features, which combined excessive laughter, a very happy disposition, hyperactivity, a short attention span, the mouthing of objects, tantrums and stereotyped movements that gave a behavioural profile mimicking Angelman syndrome. Both patients had an increased succinyladenosine/SAICAr ratio of 1.6, and exhibited a novel homozygous missense mutation (c.674T>C; p.Met225Thr) in the exon 6 of the ADSL gene. We suggest that these clinical features might be a new presentation of adenylosuccinate lyase deficiency. On the basis of this observation, although adenylosuccinate lyase deficiency is a rare disorder, this diagnosis should be considered in patients with mental retardation and a behavioural profile suggestive of Angelman syndrome.

Adenylosuccinate lyase deficiency--first British case.

A deficiency of adenylosuccinate lyase (ASDL) is characterised by the accumulation of SAICAriboside (SAICAr) and succinyladenosine (S-Ado) in body fluids. The severity of the clinical presentation correlates with a low S-Ado/SAICAr ratio in body fluids. We report the first British case of ADSL deficiency. The patient presented at 14 days with a progressive neonatal encephalopathy and seizures. There was marked axial and peripheral hypotonia. Brain MRI showed widespread white matter changes. She died at 4 weeks of age. Concentrations of SAICAr and SAdo were markedly elevated in urine, plasma and CSF and the SAdo/SAICAr ratio was low, consistent with the severe phenotype. The patient was compound heterozygous for 2 novel ADSL mutations; c.9 G>C (A3P) and c.572 C>T (R190X).

The effect of folic acid and folinic acid supplements on purine metabolism in methotrexate-treated rheumatoid arthritis.

OBJECTIVE: To determine if folinic acid supplementation during methotrexate (MTX) therapy for rheumatoid arthritis (RA) reduces both urinary 5-aminoimidazole-4-carboxamide (AICA) and urinary adenosine excretion more than does folic acid supplementation. AICA and adenosine are markers for MTX interference with purine metabolism. METHODS: Forty patients with RA who received MTX for 6 weeks were randomized to receive either daily folic acid or folinic acid supplements during an additional week of MTX therapy. Colorimetric and radioimmunocompetition assays were used to measure 24-hour urinary AICA and adenosine excretion levels, respectively. RESULTS: At the end of 6 weeks, 24-hour urinary levels of AICA, but not adenosine, were elevated as compared with baseline levels (i.e., prior to MTX therapy). Folinic acid, but not folic acid, supplementation normalized urinary AICA levels during MTX therapy. Relatively high urinary levels of AICA were correlated with reduced disease activity. No similar correlations were seen with urinary adenosine levels. CONCLUSION: The blockade of purine nucleotide biosynthesis by MTX at the AICA ribonucleotide transformylase-catalyzed step may be related to the efficacy of MTX, and this blockade is effectively relieved by folinic acid, but not by folic acid, supplementation.

Urinary adenosine and aminoimidazolecarboxamide excretion in methotrexate-treated patients with psoriasis.

BACKGROUND: We hypothesized that low-dose methotrexate treatment for patients with psoriasis would block purine biosynthesis at the step catalyzed by aminoimidazolecarboxamide (AICA) ribotide transformylase and would inhibit adenosine metabolism as evidenced by increased urinary levels of AICA and adenosine, respectively. Eight patients collected a 24-hour urine specimen on the day before their methotrexate dose and the next day during their methotrexate dose. Eight age- and sex-matched controls also collected a 24-hour urine sample. Urinary AICA and adenosine were assayed by spectrophotometric and radioimmune assays, respectively; means are reported as micromole per millimole of creatinine and were compared by the paired t test (1-tailed). OBSERVATIONS: Mean AICA excretion increased from 1.30 micromol/mmol on the day before to 1.85 micromol/mmol on the day during methotrexate dosing (P<.01). Mean adenosine values increased from 0.68 to 1.07 micromol/mmol, (P<.03). Controls had mean AICA and adenosine levels of 1.29 and 0.50 micromol/mmol, respectively. During the day of methotrexate dosing, patients had higher mean AICA and adenosine levels when compared with controls (P<.01). Mean AICA levels increased from 1.36 to 2.06 micromol/mmol (P<.025), and mean adenosine levels increased from 0.72 to 1.25 micromol/mmol (P<.025) in 5 patients showing improvement in clinical disease activity. In contrast, 3 patients with no change or worsening in clinical disease activity had smaller increases. CONCLUSIONS: Methotrexate treatment of patients with psoriasis inhibits AICA ribotide transformylase and adenosine metabolism. Since adenosine is a T-lymphocyte toxin, it may be partially responsible for the immunosuppressive effect.

Adenylosuccinase deficiency with neonatal onset severe epileptic seizures and sudden death.

We report a male infant with adenylosuccinase deficiency who developed epileptic seizures on the second day of life. Growth was normal and seizures were well controlled with anti-epileptic drugs. Despite axial hypotonia associated with peripheral hypertonicity he presented some development until seven months of age, when he developed high fever and died within a few hours. Although clinical heterogeneity in this disorder of purine synthesis and interconversion is well-known, in 14 out of 17 cases who experienced epilepsy seizures started after the first year of life. The early presentation in our index patient followed by his sudden death at the age of 7 months has not been described before. A search for disorders of purine metabolism should be included in the screening programme for every child with severe neonatal convulsions.

New methods for detection of bladder cancer.

Superficial bladder cancer is characterized by a high incidence of recurrence and a low risk of progression. Cystoscopy has been the mainstay for bladder cancer detection with additional information provided by urine cytology. Several new markers, including the BTA series of markers, NMP22 and FDP, have been approved for clinical use; numerous others continue to be evaluated. Markers help to detect clinically occult bladder cancer and to increase the interval of cystoscopic evaluation. The former indication emphasizes specificity (if a marker has high specificity, the marker-directed biopsies are frequently positive) and the latter, sensitivity (if a marker has high sensitivity, there is a low risk of deferring cystoscopy when bladder cancer is present). Because no marker or combination of markers has 100% sensitivity and 100% specificity, the selection of markers for clinical use rests on the desired objective and the performance characteristics of the available assays.

Detection of 5'-phosphoribosyl-4-(N-succinylcarboxamide)-5-aminoimidazole in urine by use of the Bratton-Marshall reaction: identification of patients deficient in adenylosuccinate lyase activity.

The Bratton-Marshall reaction can be used to identify patients with adenylosuccinate lyase deficiency. These patients excrete in their urine the dephosphorylated derivative of the de novo purine synthesis intermediate 5'-phosphoribosyl-4-(N-succinylcarboxamide)-5-aminoimidazole (SAICAR). The test described here depends on a coupling reaction of N-1-naphthylethylenediamine with diazotized ribosyl-4-(N-succinylcarboxamide)-5-aminoimidazole giving rise to a fast developing purple chromaphore with a maximum absorbance at 555 nm. Using the closely related compound ribosyl-5-amino-4-imidazolecarboxamide (AICA riboside) as a standard, concentrations as low as 1.0 microM produce a visible color change. The absorption at 555 nM of the azo compound increases as a linear function of the concentration of AICA riboside in the reaction. The use of a filter-paper dipstick for urine sampling and storage is also described. The two metabolites which are present in increased concentration in biological fluids of adenylosuccinate lyase deficient patients are stable on the dipstick for at least 60 days when stored at room temperature (25 degrees C).