Native Urine Research Articles

Acidification is required for calcium and magnesium concentration measurements in equine urine

BackgroundAcidification of equine urine to promote dissociation of ion complexes is a common practice for urine ion concentration measurements. The objective of this study was to evaluate the effect of acidification and storage after acidification on calcium (Ca), magnesium (Mg) and phosphate (P) concentrations and on fractional excretion (FE) of these electrolytes. Thirty-two fresh equine urine samples were analysed between December 2016 and July 2020. Complete urinalysis (stick and sediment) was performed on all samples. Ca, Mg, P and creatinine concentrations were measured in supernatant of centrifuged native urine, urine directly centrifuged after acidification and urine centrifuged 1 hour after acidification. Urine was acidified with hydrochloric acid to reach a pH of 1–2. Ca, Mg, P and creatinine concentrations were also measured in blood plasma, and fractional excretion of each electrolyte was calculated. Equality of medians was tested with Friedman tests and Bland-Altman bias plots were used to show the agreement between conditions.ResultsAcidification had a statistically significant effect on Ca and Mg concentrations, FECa and FEMg. Bland-Altman plot revealed a strong positive proportional bias between Ca concentration in native and acidified urine with a mean bias of 17.6 mmol/l. For Mg concentration, the difference between native and acidified urine was small with a mean bias of 1.8 mmol/l. The increase in FECa was clinically relevant. Storage of acidified urine had no effect on any of the measured ion concentrations. All P concentrations in native urine samples were below the detection limit of the assay and statistical analysis and calculation of FEP was not possible.ConclusionsUrine acidification is essential for accurate measurement of Ca and Mg concentrations and therefore FE calculations in equine urine. Storage time of 1 hour after acidification does not significantly change Ca and Mg concentrations.

LC-MS/(MS) confirmatory doping control analysis of intact phase II metabolites of methenolone and mesterolone after Girard's Reagent T derivatization.

In the present study, the application and evaluation of Girard's Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid-liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis.

Diagnosis of latent forms of renal pathology by the structure of the solid phase of urine

Introduction. Currently, chronic kidney disease is called a "silent killer", since patients with the development of a pathological process do not experience pain for a long time, modern functional and laboratory diagnostic methods also do not allow to determine the early signs of this disease, which contributes to the chronicity of the disease. Purpose: to reveal the hidden signs of the pathology of the urinary system according to the structures of the solid phase of the patient's urine. Materials and methods. The urine of 359 patients without signs of renal pathology was studied. The method of analysis of solid-phase structures of dehydrated urine (facies) was applied by microscopy in ordinary and polarized light. Processing the structures of the solid phase of urine with a stream of steam of boiling water caused their recrystallization, which made it possible to reveal hidden markers of renal pathology. Results. Analysis of the structure of native urine facies (N-facies) showed that in all examined patients they were completely covered with salt crystals, which corresponded to the physiological state of the body. Steam jet treatment of urine N-facies revealed organic (non-mineralized) inclusions in 23 patients, and a marker of bacteriuria was detected in 9 patients. The study of urine facies with the Lithos protein reagent (LR-facies) showed that the Lithos-reagent protein dissolves organic inclusions and they cease to appear in LR-facies. In 15 patients, organo-mineral microaggregates were detected in the marginal zone of the LR-facies of urine. After exposure to steam, these microaggregates disappeared in 7 patients, and remained in 8. In 9 patients, in whose N-facies, after steaming, a marker of bacteriuria was manifested, in the LR-facies of urine of 7 patients, a marker of candidiasis was detected. Conclusion. The study of the structure of urine facies before and after their treatment with boiling water steam makes it possible to identify hidden forms of renal pathology: insufficient filtration capacity of the kidneys, the risk of nephrolithiasis, bacterial and fungal infection. The data obtained make it possible to ensure timely prevention and treatment in the early stages of the disease.

Metabolic Molecular Diagnosis of Inflammatory Bowel Disease by Synergistical Promotion of Layered Titania Nanosheets with Graphitized Carbon.

The online version contains supplementary material available at 10.1007/s43657-022-00055-0.

Assessment of urine sample quality by the simultaneous measurement of urinary γ-glutamyltransferase and lactate dehydrogenase enzyme activities: possible application to unravel cheating in drugs of abuse testing.

Evaluation of the simultaneous measurement of urinary γ-glutamyltransferase (γGT) and lactate dehydrogenase (LDH) to discriminate fresh from previously frozen specimens in urine drug monitoring. Two widely available photometric tests (Siemens Healthineers Atellica) were used to determine the range of urinary γGT and LDH excretion and to study the decay in urinary enzyme activity under various storage conditions (room temperature, 4-8°C,-18°C,-80°C). From these data, cut-off values were established and evaluated in split (fresh/frozen) specimens. Both assays allow robust, reliable, and simultaneous determination of urinary γGT and LDH. In healthy subjects, the 95% reference intervals for enzyme activity in native urine were γGT: 24.4-100.4U/g Crea (creatinine) and LDH: 2.5-45.8U/g Crea. Frozen storage for at least 7days at-18°C resulted in a loss of activity to less than 50% in both enzymes. Cut-offs for frozen samples were γGT≤33.2U/g Crea and LDH≤ 8.4U/g Crea. When applied to 100 sample pairs (fresh/frozen), 86.5% (173/200) of the measurements were conclusive and the combination of concordant enzyme measurements (low γGT/low LDH or high γGT/high LDH) was able to predict the mode of storage with a sensitivity of 96.3% and a specificity of 96.7%. The additional measurements of urinary γGT and LDH can be used to detect previously frozen urine specimens. A simple protocol is proposed to provide additional information on sample quality when deceit is suspected. The procedure can be easily integrated into the standard workflow of urinary drug monitoring.

Application of photodynamic inactivation against pathogens of urinary tract infections

Photodynamic inactivation (PDI) is an alternative to antibiotic therapy method for biocidal action against microorganisms, which can be used for lithotripsy and sanitation of the bladder cavities. Objective. Selection of parameters and application PDI against uropathogenic microorganisms. Materials and Methods. In this study we used bacterial strains isolated from urine samples of patients. Differentiation media and biochemical plates were used for identification of microorganisms. The sensitivity of uropathogenic microorganisms to PDI was studied on pure cultures and in native urine. The photosensitizer “Photoditazine” (50 µg/ml) was used in the work, as well as Triton X-100 (5 % vol.) was applying to increase the permeability of the cell wall of gram-negative microorganisms. The samples were irradiated by a medical laser device “Latus-K” with a wavelength of 662 nm. To assess the effectiveness of PDI, the values of the logarithmic decrease of colony-forming unit (CFU) of the microorganisms were calculated. Statistical analysis was made by Statistica 10.0 and Mann-Whitney criterion. Results. 50 strains of uropathogens belonging to 18 species were isolated from 36 samples of native urine. Among them, the most common were S. aureus, E. coli, P. aeruginosa, K. pneumoniae. The value of logarithmic decrease in CFU for gram-positive bacteria ranged from 5 to 6, which corresponds to inactivation 99.999-99.9999% of bacterial cells in a sample. For gram-negative strains, this value was slightly lower and ranged from 4 to 5.5, which, nevertheless, corresponds to inactivation 99.99-99.999% of CFU bacteria. The addition of Triton X-100 increase the efficiency from 46% to 99.99% for E. coli, from 99% to 99.99% for P. mirabilis, from 16% to 94% for K. pneumoniae and from 97% to 99.999% for P. aeruginosa. It should be noted that the PDI was affect microorganisms both in isolated pure cultures and in native urine. Conclusions. Photodynamic inactivation may be considered as an alternative to antibiotic therapy method of biocidal action against uropathogenic microorganisms.

Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes.

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.

Development, validation of a GC–MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Уромодулин: связь олигомерных форм и функции

Введение. Уромодулин является основным белком присутствующим в моче в норме, его физиологическая роль очень разнообразна. Оценка его вклада в стабилизацию коллоида мочи в норме и при различных патологических состояниях требует детального исследования олигомерных форм, присутствующих в моче, их структуры и функций. Цель работы - изучение структурных особенностей олигомерных форм белка уромодулина в моче здоровых добровольцев и пациентов с подтверждённым уролитиазом и выявление связи структуры белка и его роли в стабилизации коллоида мочи. Методика. Методом динамического рассеяния света, анализом треков наночастиц и измерением дзета-потенциала изучены биофизические свойства изоформ уромодулина (UM), присутствующего в нативной моче в виде олигомерных форм, из которых можно выделить 2 основные: UM(7) - глобулярная молекула массой 7MDa, характеризуется гидродинамическим радиусом Rh=90-100 нм и отрицательным поверхностным зарядом величиной 25 - 30 мВ; UM(28) - массой 28MDa обладает палочкоподобной структурой с гидродинамическим радиусом Rh=200-300 нм и существенно меньшим по величине поверхностным зарядом 0 - -7 мВ. Результаты. В норме в моче UM(7) является доминантной формой, при этом вклад UM(28) либо отсутствует, либо незначителен. При уролитиазе доля UM(7) радикально уменьшается и вклад UM(28) становится основным. В модельных экспериментах показаны различия этих переходов в моче здоровых лиц и пациентов с уролитиазом в зависимости от величины pH и концентрации одновалентных катионов: натрия, калия и аммония. Заключение. На основании полученных данных существенно расширено представление о саногенетической системе коллоидного гомеостаза мочеобразования и патогенезе кристаллогенеза. Аппроксимация выдвинутой концепции развития патологического кристаллогенеза в клиническую практику расширяют информативность превентивной диагностики уролитиаза. Introduction. Uromodulin is the major protein, which is normally present in urine and plays multiple physiological roles. Evaluation of the uromodulin contribution to stabilization of urinary colloids in normal and various pathological conditions requires a comprehensive study of uromodulin oligomeric forms occurring in urine, their structure and functions. The aim of this work was studying structural features of uromodulin oligomeric forms in the urine of healthy volunteers and patients with confirmed urolithiasis and identifying a relationship between the protein structure and role in stabilization of urinary colloids. Methods. Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and measurement of zeta potential were used to study biophysical properties of uromodulin (UM) isoforms. UM is present in native urine as oligomeric forms, including two major ones: i) UM (7), a 7MDa globular molecule characterized by a hydrodynamic radius Rh = 90-100 nm and a negative surface charge of 25-30 mV and ii) UM (28), a rod-like 28MDa molecule with a hydrodynamic radius of Rh=200-300 nm and a significantly lower surface charge of 0 --7 mV. Results. Normally, UM (7) is a dominant form in urine whereas the UM (28) contribution is either non-existent or minor. In urolithiasis, the proportion of UM (7) decreases drastically, and the contribution of UM (28) becomes primary. Model experiments showed differences between these transitions in the urine of healthy individuals and patients with urolithiasis depending on pH values and concentrations of monovalent cations, including sodium, potassium, and ammonium. Conclusion. The study results considerably expanded the concept of the sanogenetic system of colloidal homeostasis in urine formation and the pathogenesis of crystallogenesis. Approximating the proposed concept of pathological crystallogenesis in clinical practice expands the informative value of preventive diagnosis of urolithiasis.

Optimization of artificial urine formula for in vitro cellular study compared with native urine.

Several artificial urine (AU) formulas have been developed to mimic the normal urine. Most of them are protein-free, particularly when secreted proteins (secretome) is to be analyzed. However, the normal urine actually contains a tiny amount of proteins. We hypothesized that urinary proteins at physiologic level play a role in preservation of renal cell biology and function. This study evaluated the effects from supplementation of 0-10% fetal bovine serum (FBS) into the well-established AU-Siriraj protocol on MDCK renal tubular cells. Time to deformation (TD) was reduced by both native urine and AU-Siriraj without/with FBS compared with complete culture medium (control). Among the native urine and AU-Siriraj without/with FBS, the cells in AU-Siriraj+2.5% FBS had the longest TD. Supplementation of FBS increased cell death in a dose-dependent manner (but still <10%). Transepithelial electrical resistance (TER) of the polarized cells in the native urine was comparable to the control, whereas that of the cells in AU-Siriraj+2.5% FBS had the highest TER. These data indicate that supplementation of 2.5% FBS into AU-Siriraj can prolong time to deformation and enhance polarization of renal tubular cells. Therefore, AU-Siriraj+2.5% FBS is highly recommended for in vitro study of cell biology and function (when secretome is not subjected to analysis).

Identification of pathogens from native urine samples by MALDI-TOF/TOF tandem mass spectrometry

BackgroundReliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample.MethodsIn total, 16 urine samples having more than 105 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested.ResultsHere we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing.ConclusionsOur findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

Establishment of a reference procedure to measure urine-formed elements and evaluation of an automated urine analyzer

A standardized reference method is needed to accurately and precisely measure urine-formed elements (UFEs; red blood cells [RBCs], white blood cells [WBCs], and squamous epithelial cells [sECs]). We compared the results from a standard method with those from an automated analyzer. Trained technicians used standardized bright-field microscopy of fresh non-centrifuged urine samples, and disposable 1 µl chambers. Fifteen experienced technicians from 5 hospitals (3 per hospital) each performed 6 manual counts of 10 different native urine samples using a manual chamber and standard methods. The sEC counts were at least 50/µL, and the coefficient of variation (CV) was less than 14%; the RBC and WBC counts were at least 200/µL and the CVs were less than 7%. The same samples were also analyzed 6 times using automated analyzers. The means, CVs, and biases were determined. The median CVs for the manual measurements were 6.4% (WBCs), 6.6% (RBCs), and 12.7% (sECs). The CVs of the automated analyzer were 4.7% (WBCs), 5.6% (RBCs), and 9.2% (sECs). Biases between the automated and manual methods were −2.9% to 5.0%(WBCs), −0.8% to 8.8% (RBCs) and −2.8% to 9.4% (sECs). The count mean values and expanded uncertainties of these counts were (224.5 ± 15.0) cells/µL, (234.2 ± 16.2) cells/µL, and (61.5 ± 7.9) cells/µL, respectively. The standardized manual method for measuring UFEs had high precision and accuracy, making it a suitable reference method. Use of this reference method to calibrate an automated analyzer improved the accuracy of automated analysis.

The standardization of the report for urine cell counting-A converting factor for Sysmex UF-1000i.

BackgroundMulticenter laboratory may apply both automated flow cytometer and microscopy for urinalysis. Automated flow cytometer such as Sysmex UF‐1000i evaluates particles with native urine without centrifugation and reports as “counts per μL.” Microscopic examination recommended as the reference method for urine sediment analysis reports results as “counts per HPF (or μL).” Moreover, some results from flow cytometer are needed to be checked visually under microscopy. Therefore, it is worth to establish the consistency of the results from these two methods.MethodsUrine specimens from 412 patients were examined with Sysmex UF‐1000i and manual microscopy using FAST‐READ disposable counting chambers. White blood cell (WBC) and red blood cell (RBC) counting results from UF‐1000i after transferred with the converting factor (0.297) we estimated were compared with that from microscopic examination. Method comparison was performed using Passing‐Bablok analysis.ResultsAfter transferred with the converting factor (0.297), cell counting results from UF‐1000i showed a good correlation with that derived by the reference method (R 2 was 0.868 for RBCs (P < 0.001), 0.882 for WBCs (P < 0.001)). Passing‐Bablok analysis showed no systematic difference (intercept estimate, −1 [95%CI, −7 to 3] and slightly proportional (slope estimate, 1.2 [95%CI, 1.0 to 1.7]) bias between concentrations of cells measured by manual microscopy and Sysmex UF‐1000i using the converting factor.ConclusionThe converting factor (0.297) helps to transfer “counts per μL (non‐centrifugal urine)” to “counts per μL (equal to centrifugal urine),” and to keep the urine particle analysis results of Sysmex UF‐1000i consistent with the results from the reference method.

Cervical cancer detection by DNA methylation analysis in urine

Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (r = 0.508–0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744–0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Urinary Lipidomics: evidence for multiple sources and sexual dimorphism in healthy individuals.

Urinary lipidomics may add new valuable biomarkers to the diagnostic armamentarium for early detection of metabolic and kidney diseases. Sources and composition of urinary lipids in healthy individuals, however, have not been investigated in detail. Shotgun lipidomics was used to quantify lipidomic profiles in native urine samples from 16 individuals (eight men, eight women) collected in five fractions over 24 h. All probands were comprehensively characterized by urinary and clinical indices. The mean total urinary lipid concentration per sample was 0.84 μM in men and 1.03 μM in women. We observed significant intra- and interindividual variations of lipid concentrations over time, but failed to detect a clear circadian pattern. Based on quantity and subclass composition it seems very unlikely that plasma serves as major source for the urinary lipidome. Considering lipid metabolites occurring in at least 20% of all samples 38 lipid species from 7 lipid classes were identified. Four phosphatidylserine and one phosphatidylethanolamine ether species (PE-O 36:5) were detectable in almost all urine samples. Sexual dimorphism has been found mainly for phosphatidylcholines and phosphatidylethanolamines. In men and in women urinary lipid species were highly correlated with urinary creatinine and albumin excretion, reflecting glomerular filtration and tubular transport processes. In women, however, lipid species deriving from urinary cells and cellular constituents of the lower genitourinary tract considerably contributed to the urinary lipidome. In conclusion, our study revealed the potential of urinary lipidomics but also the complexity of methodological challenges which have to be overcome for its implementation as a routine diagnostic tool for renal, urological and metabolic diseases.

Duchenne Muscular Dystrophy Myogenic Cells from Urine-Derived Stem Cells Recapitulate the Dystrophin Genotype and Phenotype.

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization.

Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation.In this study, a novel multi-target approach based on liquid chromatography and high resolution/high accuracy mass spectrometry is presented to screen for more than 200 analytes of various classes of doping agents far below the required detection limits in sports drug testing. Here, classic groups of drugs as diuretics, stimulants, β2-agonists, narcotics and anabolic androgenic steroids as well as various newer target compounds like hypoxia-inducible factor (HIF) stabilizers, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), plasma volume expanders and other doping related compounds, listed in the 2016 WADA prohibited list were implemented. As a main achievement, growth hormone releasing peptides could be implemented, which chemically belong to the group of small peptides (<2kDa) and are commonly determined by laborious and time-consuming stand-alone assays.The assay was fully validated for qualitative purposes considering the parameters specificity, robustness (rRT: <2%), intra- (CV: 1.7–18.4 %) and inter-day precision (CV: 2.3–18.3%) at three concentration levels, linearity (R2>0.99), limit of detection (0.1–25ng/mL; 3′OH-stanozolol glucuronide: 50pg/mL; dextran/HES: 10μg/mL) and matrix effects.

Measurement of unlabeled and stable isotope-labeled homoarginine, arginine and their metabolites in biological samples by GC-MS and GC-MS/MS.

Circulating and excretory L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA) are cardiovascular risk factors. L-Arginine (Arg) is the common precursor of hArg and ADMA. This protocol describes gas chromatography-mass spectrometry (GC-MS) and gas chromatography-mass spectrometry-mass spectrometry (GC-MS/MS) methods for the quantitative determination of hArg, Arg and ADMA in biological samples, including human plasma, urine and sputum. Aliquots (10 µL) of native urine, plasma or serum ultrafiltrate (cutoff, 10 kDa), and acetone-deproteinized sputum samples are evaporated to dryness. Then, amino acids are derivatized to their methyl ester N-pentafluoropropionyl derivatives. In parallel, trideuteromethyl ester N-pentafluoropropionyl derivatives of hArg, Arg and ADMA are de novo synthesized from the unlabelled amino acids and used as internal standards. Alternatively, commercially available stable isotope-labeled analogs of hArg, Arg and ADMA are used as internal standards, and they are added to the native biological samples. Quantification is performed by selected ion monitoring in GC-MS and selected reaction monitoring in GC-MS/MS. By these protocols, unlabelled and stable isotope-labeled hArg, Arg and their metabolites including ADMA and ornithine can be measured equally accurately and precisely by GC-MS and GC-MS/MS in several different biological fluids in experimental and clinical settings.

Protein composition of human urine during five-day immersion

Dynamics of protein spectrum (12 groups of proteins) of the native daily urine was studied by gradient polyacrylamide gel electrophoresis in the experiment on five-day dry immersion in 14 subjects aged from 19 to 26 years. It was established that changes in the level of urinary protein excretion were not observed during the experiment. Nevertheless, the tendency towards formation of a number of shifts in the urinary protein spectrum was found. The excretion of low-molecular-weight proteins was decreased and that of albumin was increased, which reflects the development of tubular reabsorption remodulation under the influence of experimental exposure. The level of Tamm-Horsfall protein was steadily increased, which is likely to be a protective reaction of the body. An incremental reduction of immunoglobulin content in the urine was observed, which is probably related to decreasing permeability of the glomerular filter for high-molecular-weight proteins.