A toxicokinetic model to assess exposure to folpet fungicide from urinary biomarkers

Folpet is one of the most widely employed fungicides in agriculture. It is typically used in the culture of vegetables, fruits and ornamental plants. Once absorbed in the human body, it has been found to be very reactive, especially in acid conditions. According to various in vitro and in vivo experiments in animals, Folpet is first fractioned at the N-S link when in contact with aqueous solutions and thiol groups. From this non-enzymatic process a phthalimide (PI) molecule is formed, which may be used as a biomarker of exposure, along with the short-lived thiophosgene. We have built a human toxicokinetic model to account for the biotransformation of Folpet into PI and its subsequent excretion while accounting for other non-monitored metabolites. The mathematical parameters of the model were determined accordingly from best-fits to the time courses of PI in blood and urine of five volunteers administered orally 1 mg/kg and dermally 10 mg/kg of Folpet. In both cases, the mean elimination half-life of PI from the body (either through faeces, urine or metabolism) was found to be 31.6 h. The average final fractions of administered dose recovered in urine as PI were 0.025% and 0.002%, for oral and dermal administration, respectively after 96 h. According to the model, when orally administered, PI rapidly hydrolyzes to phthalamic and phthalic acids such that only 0.04% of the PI found in the gastrointestinal tract is absorbed into the blood stream. Likewise, after dermal application, model predicts that only 7.4% of the applied Folpet dose crosses the epidermis. In the model, the PI initial metabolite of Folpet is formed in the dermis and further metabolized prior to reaching systemic circulation, such that only 0.125% of PI formed at the site-of-entry reaches systemic blood. Our mathematical model is in accordance with both measures of blood (R2=0.57 for dermal and R2=0.66 for oral) and urine (R2 =0.98 for dermal and R2=0.99 for oral).

Factors associated with 24-hour urinary volume: the Swiss salt survey.

BACKGROUND: Low 24-hour urine volume (24 UV) may be a significant risk factor for decline in kidney function. We therefore aimed to study associated markers and possible determinants of 24 UV in a sample of the Swiss population. METHODS: The cross-sectional Swiss Salt Study included a population-based sample of 1535 (746 men and 789 women) individuals from three linguistic regions of Switzerland. Data from 1300 subjects were available for the present analysis. 24 UV was measured using 24-hour urine collection. Determinants of 24 UV were identified using multivariable linear regression models. RESULTS: In bivariate analysis, 24 UV was higher in women compared to men (2000 ml/24 h [interquartile range (IQR): 1354, 2562] versus 1780 ml/24 h [IQR: 1244, 2360], p = 0.002). In multivariable regression analyses, independent associated markers of 24 UV were female sex (β = 280, 95% confidence interval [CI]: 174, 386, p < 0.0001), fluid intake (β = 604, 95% CI: 539, 670, p < 0.0001), sodium excretion (β = 4.2, 95% CI: 3.4, 4.9, p < 0.0001) age (β = 6.6, CI: 3.4, 9.7, p < .0001), creatinine clearance (β = 2.4, CI: 0.2, 4.6, p = 0.04), living in the German-speaking part of Switzerland (β = 124, CI: 29, 219, p = 0.01), alcohol consumption (β = 41, CI: 9, 73, p = 0.01 for increasing categories of alcohol consumption), body mass index (β = -32, CI: -45, -18, p < 0.0001), current smoking (β = -146, CI: -265, -26, p = 0.02), and consumption of meat and cold cut (β = -56, CI: -108, -5, p = 0.03). CONCLUSION: In this large population-based, cross-sectional study, we found several strong and independent correlates for 24 UV. These findings may be important to improve our understanding in the development of chronic kidney disease.

Urinary oxalate and urate to creatinine ratios in a healthy pediatric population.

The purpose of the study was to determine reference percentiles for the urinary (U) oxalate (Ox) and urate (Ura) to creatinine (Cr) concentration ratios in the second morning urine of healthy infants, children, and adolescents. The urinary oxalate and urate to creatinine ratios were determined in the spontaneously voided second morning urine sample. To test reproducibility, two urine samples were analyzed on 2 consecutive weeks in 63% of the subjects. Three hundred eighty-four healthy children (181 girls, 203 boys), aged 1 month to 17 years, from nurseries, kindergartens, and schools of Lausanne, Switzerland, were studied. The 5th and 95th percentiles were determined from the total number of urine samples (627) after confirmation that there was no order effect between repeated measurements and there were no significant sex differences. A nonlinear regression analysis in terms of age was used to smooth the calculated percentiles. In this manner, curves were obtained from which the reference values can be read at any given age. The 95th percentiles decreased with age: for UOx/Cr from 0.175 mg/mg (0.22 mol/mol) at 1 to 6 months to 0.048 mg/mg (0.06 mol/mol) from 7 years and beyond; and UUra/Cr from 2.378 mg/mg (1.6 mol/mol) at 1 to 6 months to 0.594 mg/mg (0.4 mol/mol) in adolescence. We provide 5th and 95th percentile curves for the UOx/Cr and UUra/Cr ratios determined from the second morning urine samples in a large cohort of healthy infants, children, and adolescents. Values were determined by standard analytical chemical techniques and were analyzed by powerful statistical methods. The calculated 95th percentile for the UOx/Cr values fell rather rapidly and reached normal adult values by the age of 7 years, whereas for UUra/Cr, the 95th percentile decreased slowly and stabilized in adolescence.

In vitro growth of human urinary tract smooth muscle cells on laminin and collagen type I-coated membranes under static and dynamic conditions.

This study investigates in vitro growth of human urinary tract smooth muscle cells under static conditions and mechanical stimulation. The cells were cultured on collagen type I- and laminin-coated silicon membranes. Using a Flexcell device for mechanical stimulation, a cyclic strain of 0-20% was applied in a strain-stress-time model (stretch, 104 min relaxation, 15 s), imitating physiological bladder filling and voiding. Cell proliferation and alpha-actin, calponin, and caldesmon phenotype marker expression were analyzed. Nonstretched cells showed significant better growth on laminin during the first 8 days, thereafter becoming comparable to cells grown on collagen type I. Cyclic strain significantly reduced cell growth on both surfaces; however, better growth was observed on laminin. Neither the type of surface nor mechanical stimulation influenced the expression pattern of phenotype markers; alpha-actin was predominantly expressed. Coating with the extracellular matrix protein laminin improved in vitro growth of human urinary tract smooth muscle cells.

The rapid urinary drug toxicology screen in the emergency room: a useful tool ?

Intoxications are a frequent problem in the ER. In the vast majorityof cases, supportive treatment is sufficient. Severe intoxications withunknown agents are considered an indication for a urinary drug screen,and are recommended by several toxicology centers. However, theirusefulness for patient management remains uncertain.Study objectives: Evaluation of the impact of a urinary drug screen(Biosite Triage TOX Drug Screen) testing 11 substances(acetaminophen, amphetamines, methamphetamines, barbiturates,benzodiazepines, cocaïne, methadone, opioids, phencyclidine,cannabis, tricyclic antidepressants) on initial adult patient managementin the emergency department of a university hospital with ~35.000annual admissions.Methods: Observational retrospective analysis of all tests performedbetween 09/2009 and 09/2010. A test utility was defined as useful if itresulted in the administration of a specific antidote (Flumazenil/Naloxone), the use of a quantitative confirmatory toxicologic test, or achange in patient's disposition.Results: 57 tests were performed. Patient age was 32 ± 11 (SD) years;58% were men; 30% were also intoxicated with alcohol. Two patientsdied (3.5%): the first one of a diphenhydramin overdose, the other of ahypertensive intracerebral hemorrhage believed to be caused cocaineabuse but a negative urine test. Test indications were: 54% firstpsychotic episode; 25% acute respiratory failure; 18% coma; 12%seizure; 11% opioids toxidrome; 7% sympathicomimetic toxidrome; 5%hypotension; 4% ventricular arrhythmia (VT, VF, torsades de pointes)or long QT. 75% of tests were positives for >=1 substance (mean 1.7 ±0.9). 47% of results were unexpected by history. 18% of resultsinfluenced patient management: 7% had a negative test that confirmedthe diagnosis of endogenous psychosis in a first psychotic episode, andallowed transfer to psychiatry; 5% received flumazenil/naloxone;2% had an acetaminophen blood level after a positive screen; finally,4% had an unexpected methadone abuse that required prolongationof hospital stay.Conclusions: A rapid urinary toxicologic screen was seldom used inour emergency department, and its impact on patient managementwas marginal: only one in 6 tests influenced treatment decisions.

Effects of neuropeptide Y on intrarenal hemodynamics, plasma renin activity and urinary sodium excretion in rats.

Neuropeptide Y (NPY) is a vasoconstrictor peptide possibly involved in the regulation of renal sodium handling and renin release. This investigation was undertaken to assess in conscious normotensive rats the acute effects of a non-pressor dose of NPY on renal plasma flow, glomerular filtration rate, sodium excretion and plasma renin activity. Experiments were also performed during concomitant beta-adrenoceptor stimulation with isoproterenol. NPY per se had no effect on the studied parameters. Renal plasma flow was increased by isoproterenol and was significantly higher when the beta-adrenoceptor stimulant was infused alone (13.4 +/- 2.1 ml/min, p < 0.05, mean +/- SEM) that when administered together with NPY (7.2 +/- 2.0 ml/min). This was also true for glomerular filtration rate (3.3 +/- 0.3 vs. 1.8 +/- 0.3 ml/min, p < 0.01) and plasma renin activity (6.3 +/- 1.7 vs. 2.1 +/- 0.4 ng Ang I/ml/h, p < 0.05). Our data however do not allow to deduce whether the inhibitory effect of NPY on isoproterenol-induced renin release is mediated by changes in intrarenal hemodynamics or a direct effect on juxtaglomerular cells.

Urinary sphincter based on electronics and artificial muscles

Objectives: The AMS 800TM is the current artificial urinary sphincter (AUS) for incontinence due to intrinsic sphincter deficiency. Despite good clinical results, technical failures inherent to the hydraulic mechanism or urethral ischemic injury contribute to revisions up to 60%. We are developing an electronic AUS, called ARTUS to overcome the rigors of AMS. The objective of this study was to evaluate the technical efficacy and tissue tolerance of the ARTUS system in an animal model.Methods: The ARTUS is composed by three parts: the contractile unit, a series of rings and an integrated microprocessor. The contractile unit is made of Nitinol fibers. The rings are placed around the urethra to control the flow of urine by squeezing the urethra. They work in a sequential alternative mode and are controlled by a microprocessor. In the first phase a three-rings device was used while in the second phase a two-rings ARTUS was used. The device was implanted in 14 sheep divided in two groups of six and eight animals for study purpose. The first group aimed at bladder leak point pressure (BLPP) measurement and validation of the animal model; the second group aimed at verifying mid-term tissue tolerance by explants at twelve weeks. General animal tolerance was also evaluated.Results: The ARTUS system implantation was uneventful. When the system was activated, the BLPP was measured at 1.038±0.044 bar (mean±SD). Urethral tissue analysis did not show significant morphological changes. No infection and no sign of discomfort were noted in animals at 12 weeks.Conclusions: The ARTUS proved to be effective in continence achievement in this study. Histological results support our idea that a sequential alternative mode can avoid urethral atrophy and ischemia. Further technical developments are needed to verify long-term outcome and permit human use.

Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing.

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 0/00 and maintained at this level for two weeks for two individuals. The steroids under investigation were 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol (excreted as glucuronides) and ETIO, ANDRO and 3β-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 0/00, shifts of approximately 30 0/00 were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

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

RATIONALE: AICAR (5-aminoimidazole-4-carboxamide 1β-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.

Untargeted profiling of urinary steroid metabolites after testosterone ingestion: opening new perspectives for antidoping testing.

AIM: Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. RESULTS: Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. CONCLUSION: This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.