[Urinary specific gravity--comparative measurements using reagent strips and refractometer in 340 morning urine samples]

The specific gravity of urine (SG) indicates the number and weight of solute particles in urine; its measurement is helpful in interpreting proteinuria detected by dipstick tests and in monitoring adequate hydration in patients with nephrolithiasis. Four methods for measuring SG or osmolality of urine are currently available (depression of the freezing-point, urometry, refractometry, cation exchange on a reagent strip). Using a recently developed reagent strip, we have measured SG in morning urines of 340 non-selected outpatients and compared the results with SG measurements by refractometry of the same urines. In 86.2% of all urines, a good positive correlation between SG measured by reagent strip and refractometry was noted (r = 0.913, p = 0.0001). In 13.8% of the urines, however, the SG measured by reagent strip deviated by more than +/- 5 from the value obtained by refractometry; in 90% of these urines, glucosuria (reagent strip values too low or too high), proteinuria (values too high), or bacteriuria/leukocyturia (values too low or too high) could be found. In alkaline urine (pH > 7.0), SG values obtained by reagent strip have to be corrected by +5.

Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer

In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method).

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC-ESI-MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.

Human urine certified reference material CZ 6010: creatinine and toluene metabolites (hippuric acid and o-cresol) and a benzene metabolite (phenol)

A reference material for the biological monitoring of occupational exposure to toluene, benzene and phenol was prepared. O-cresol and hippuric acid (metabolites of toluene) are used for the biological monitoring of occupational exposure to toluene. Phenol, a metabolite of benzene, is used for the biological monitoring of exposure to benzene, but phenol can of course also be used as an indicator of exposure to phenol as well. The reference material (RM) used for the determination of these metabolites was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to toluene and those taking part in an inhalation experiment. Tests for homogeneity and stability were performed by determining urine concentrations of o-cresol, hippuric acid, creatinine and phenol. To investigate the stability of the RM, the urinary concentrations of o-cresol and phenol were monitored for eighteen months using GC and HPLC, while those of hippuric acid and creatinine were followed for five and six years, respectively, using HPLC. Analysis of variance showed that the concentrations did not change. The certified concentration values (and their uncertainties) of the substances in this reference material (phenol concentration c=6.46+/-0.58 mg l(-1); o-cresol concentration c=1.17+/-0.15 mg l(-1); hippuric acid concentration c=1328+/-30 mg l(-1); creatinine concentration c=0.82+/-0.10 g l(-1)) were evaluated via the interactive statistical programme IPECA.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Pharmacokinetics of GHB and detection window in Serum and Urine after single uptake of a low dose of GLB - an Experiment with two volunteers

During the last few years γ-hydroxybutyric acid (GHB) and γ-butyrolactone (GBL) have attracted much interest as recreational drugs and knock-out drops in drug-facilitated sexual assaults. This experiment aims at getting an insight into the pharmacokinetics of GHB after intake of GBL. Therefore Two volunteers took a single dose of 1.5 ml GBL, which had been spiked to a soft drink. Assuming that GBL was completely metabolized to GHB, the corresponding amount of GHB was 2.1 g. Blood and urine samples were collected 5 h and 24 h after ingestion, respectively. Additionally, hair samples (head hair and beard hair) were taken within four to five weeks after intake of GBL. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after protein precipitation with acetonitrile. The following observations were made: spiked to a soft drink, GBL, which tastes very bitter, formed a liquid layer at the bottom of the glass, only disappearing when stirring. Both volunteers reported weak central effects after approximately 15 min, which disappeared completely half an hour later. Maximum concentrations of GHB in serum were measured after 20 min (95 µg/ml and 106 µg/ml). Already after 4-5 h the GHB concentrations in serum decreased below 1 µg/ml. In urine maximum GHB concentrations (140 µg/ml and 120 µg/ml) were measured after 1-2 h, and decreased to less than 1 µg/ml within 8-10 h. The Ratio of GHB in serum versus blood was 1.2 and 1.6

Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis.

Leptospirosis is a global zoonotic disease. Pathogenic Leptospira species, the causative agent of leptospirosis, colonize the renal tubules of chronically infected maintenance hosts such as dogs, rats and cattle. Maintenance hosts typically remain clinically asymptomatic and shed leptospires into the environment via urine. In contrast, accidental hosts such as humans can suffer severe acute forms of the disease. Infection results from direct contact with infected urine or indirectly, through contaminated water sources. In this study, a quantitative real-time PCR specific for lipL32 was designed to detect the urinary shedding of leptospires from dogs. The sensitivity and specificity of the assay was evaluated using both a panel of pathogenic Leptospira species and clinical microbial isolates, and samples of urine collected from experimentally infected rats and non-infected controls. The lower limit of detection was approximately 3 genome equivalents per reaction. The assay was applied to canine urine samples collected from local dog sanctuaries and the University Veterinary Hospital (UVH) at University College Dublin. Of 525 canine urine samples assayed, 37 were positive, indicating a prevalence of urinary shedding of leptospires of 7.05%. These results highlight the need to provide effective canine vaccination strategies and raise public health awareness.

Detection and quantification of 56 new psychoactive substances in whole blood and urine by LC-MS/MS

BACKGROUND New psychoactive substances (NPS) have become increasingly prevalent and are sold in internet shops as 'bath salts' or 'research chemicals' and comprehensive bioanalytical methods are needed for their detection. METHODOLOGY We developed and validated a method using LC and MS/MS to quantify 56 NPS in blood and urine, including amphetamine derivatives, 2C compounds, aminoindanes, cathinones, piperazines, tryptamines, dissociatives and others. Instrumentation included a Synergi Polar-RP column (Phenomenex) and a 3200 QTrap mass spectrometer (AB Sciex). Run time was 20 min. CONCLUSION A novel method is presented for the unambiguous identification and quantification of 56 NPS in blood and urine samples in clinical and forensic cases, e.g., intoxications or driving under the influence of drugs.

Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR

Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.

Prevalence and densitometric characteristics of incomplete distal renal tubular acidosis in men with recurrent calcium nephrolithiasis

The aim of this study was to assess the prevalence of incomplete distal renal tubular acidosis (idRTA) in men with recurrent calcium nephrolithiasis and its potential impact on bone mineral density. We conducted a retrospective analysis of 150 consecutive, male idiopathic recurrent calcium stone formers (RCSFs), which had originally been referred to the tertiary care stone center of the University Hospital of Berne for further metabolic evaluation. All RCSFs had been maintained on a free-choice diet while collecting two 24-h urine samples and delivered second morning urine samples after 12 h fasting. Among 12 RCSFs with a fasting urine pH >5.8, a modified 3-day ammonium chloride loading test identified idRTA in 10 patients (urine pH >5.32, idRTA group). We matched to each idRTA subject 5 control subjects from the 150 RCSFs, primary by BMI and then by age, i.e., 50 patients, without any acidification defect (non-RTA group) for comparative biochemistry and dual energy X-ray absorptiometry (DEXA) analyses. The prevalence of primary idRTA among RCSFs was 6.7% (10/150). Patients with idRTA had significantly higher 2-h fasting and 24-h urine pH (2-h urine pH: 6.6 ± 0.4 vs. 5.2 ± 0.1, p = 0.001; 24-h urine pH: 6.1 ± 0.2 vs. 5.3 ± 0.3, p = 0.001), 24-h urinary calcium excretion (7.70 ± 1.75 vs. 5.69 ± 1.73 mmol/d, p = 0.02), but significantly lower 24-h urinary urea excretion (323 ± 53 vs. 399 ± 114 mmol/d, p = 0.01), urinary citrate levels (2.32 ± 0.82 vs. 3.01 ± 0.72 mmol/d, p = 0.04) and renal phosphate threshold normalized for the glomerular filtration rate (TmPO(4)/GFR: 0.66 ± 0.17 vs. 0.82 ± 0.21, p = 0.03) compared to non-RTA patients. No significant difference in bone mineral density (BMD) was found between idRTA and non-RTA patients for the lumbar spine (LS BMD (g/cm(2)): 1.046 ± 0.245 SD vs. 1.005 ± 0.119 SD, p = 0.42) or femoral neck (FN BMD (g/cm(2)): 0.830 ± 0.135 SD vs. 0.852 ± 0.127 SD). Thus, idRTA occurs in 1 in 15 male RCSFs and should be sought in all recurrent calcium nephrolithiasis patients. Bone mineral density, however, does not appear to be significantly affected by idRTA.

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

Radiation metabolomics. 5. Identification of urinary biomarkers of ionizing radiation exposure in nonhuman primates by mass spectrometry-based metabolomics

Mass spectrometry-based metabolomics has previously demonstrated utility for identifying biomarkers of ionizing radiation exposure in cellular, mouse and rat in vivo radiation models. To provide a valuable link from small laboratory rodents to humans, γ-radiation-induced urinary biomarkers were investigated using a nonhuman primate total-body-irradiation model. Mass spectrometry-based metabolomics approaches were applied to determine whether biomarkers could be identified, as well as the previously discovered rodent biomarkers of γ radiation. Ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry analysis was carried out on a time course of clean-catch urine samples collected from nonhuman primates (n = 6 per cohort) exposed to sham, 1.0, 3.5, 6.5 or 8.5 Gy doses of (60)Co γ ray (∼0.55 Gy/min) ionizing radiation. By multivariate data analysis, 13 biomarkers of radiation were discovered: N-acetyltaurine, isethionic acid, taurine, xanthine, hypoxanthine, uric acid, creatine, creatinine, tyrosol sulfate, 3-hydroxytyrosol sulfate, tyramine sulfate, N-acetylserotonin sulfate, and adipic acid. N-Acetyltaurine, isethionic acid, and taurine had previously been identified in rats, and taurine and xanthine in mice after ionizing radiation exposure. Mass spectrometry-based metabolomics has thus successfully revealed and verified urinary biomarkers of ionizing radiation exposure in the nonhuman primate for the first time, which indicates possible mechanisms for ionizing radiation injury.

Metabolomics reveals the metabolic map of procainamide in humans and mice

Procainamide, a type I antiarrhythmic agent, is used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25-30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice as well as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.