Pyrroles as a Potential Biomarker for Oxidative Stress Disorders.

Redox imbalance or oxidative stress that results from both environmental and genetic factors is observed in patients with schizophrenia. Therefore, identifying markers of oxidative stress in the early stages of psychosis and using antioxidant treatments as an adjuvant to antipsychotics has important implications. The reaction of p-N,N-dimethylaminobenzaldehyde (DMAB) with pyrrole moieties has been well studied for well over a century for use as a marker of oxidative stress dysregulation. Throughout this time, pyrroles have been investigated with varying veracity in urine extracts to identify elevated levels in patients diagnosed with schizophrenia. Since the 1960's, various claims have been made with respect to what causes the colour change when DMAB is added to urine extracts. Whilst the substances from this reaction have not been fully elucidated, an objective look at most studies indicates that urobilinogen is likely to be one them. Urobilinogen has also been identified as a major interferent in our results. Both pyrroles and urobilinogen condense the DMAB reaction system (form condensation products) and are quite different. The urobilinogen detected in urine forms when gut microflora chemically reduces the bilirubin content of bile acids. In comparison, evidence suggests that the pyrrole fraction originates from the fragmentation of regulatory haem by reactive oxygen species (ROS) such as hydrogen peroxide and super and nitrous oxides. Clinical studies in our laboratories have established that pyrroles as a urine biomarker have specificity in detecting schizophrenia; however, caution must be applied as the readings are subject to interference by other DMAB active compounds that are present, such as urobilinogen. This review highlights the initial chemistry in isolating pyrroles and provides recommendations for standardised laboratory testing to ensure pyrroles are correctly measured and distinguished from other by-products.

Paper-Plastic Hybrid Microfluidic Device for Smartphone-Based Colorimetric Analysis of Urine.

In this work, a disposable paper-plastic hybrid microfluidic lab-on-a-chip (LOC) has been developed and successfully applied for the colorimetric measurement of urine by the smartphone-based optical platform using a "UrineAnalysis" Android app. The developed device was cost-effectively implemented as a stand-alone hybrid LOC by incorporating the paper-based conventional reagent test strip inside the plastic-based LOC microchannel. The LOC device quantitatively investigated the small volume (40 µL) of urine analytes for the colorimetric reaction of glucose, protein, pH, and red blood cell (RBC) in integration with the finger-actuating micropump. On the basis of our experiments, the conventional urine strip showed large deviation as the reaction time goes by, because dipping the strip sensor in a bottle of urine could not control the reaction volume. By integrating the strip sensor in the LOC device for urine analysis, our device significantly improves the time-dependent inconstancy of the conventional dipstick-based urine strip, and the smartphone app used for image analysis enhances the visual assessment of the test strip, which is a major user concern for the colorimetric analysis in point-of-care (POC) applications. As a result, the user-friendly LOC, which is successfully implemented in a disposable format with the smartphone-based optical platform, may be applicable as an effective tool for rapid and qualitative POC urinalysis.

Non-clinical safety assessment of Hwangryunhaedok-tang: 13-week toxicity in Crl:CD Sprague Dawley rats.

Hwangryunhaedok-tang (Huang-Lian-Jie-Du-Tang in Chinese, Oren-gedoku-to in Japanese) is a traditional herbal medicine with a long history of use for anti-inflammatory purposes. In this study, subchronic toxicity of daily oral administration of a Hwangryunhaedok-tang water extract (HHT) at 0, 250, 750, and 2000mg/kg for 13weeks was examined in rats. Mortality, clinical signs, and changes in body weight, food consumption, clinical signs, ophthalmological examination, urinalysis, hematology, serum biochemistry, gross observation, organ weight, and histopathology were monitored in accordance with Good Laboratory Practice and OECD guidelines. We found no mortality or abnormality in clinical signs, body weight, serum biochemistry, or organ weight in HHT-treated groups in either sex. However, there were significant changes in glucose, bilirubin, urobilinogen, protein (only male) in urine after 2000mg/kg/day HHT treatment for both sexes. In hematological examinations, we found a significant decreased number of red blood cells (RBC), whereas, an increased the mean corpuscular volume, number of platelets, and rate of reticulocyte (RET) after 2000mg/kg/day HHT treatment of male rats. In male and female rats, 750 and 2000mg/kg/day HHT treatment decreased the number of RBC and increased RET. Histopathological examinations revealed stomach mucosal erosion in female rats (2000mg/kg/day). No-observed-adverse-effect levels were established for 750mg/kg HHT in rats under the conditions of this study. However, other toxicological studies are necessary to evaluate the safety of HHT fully.

Assessment of urine metabolite biomarkers for the detection of S. haematobium infection in pre-school aged children in a rural community in Zimbabwe.

BACKGROUND: Early diagnosis of urogenital schistosomiasis is key to its control and elimination. The current gold standard microscopic examination techniques lack sensitivity in detecting light Schistosomiasis infections in pre-school aged children thus it is urgent to develop diagnostic tools that may be integrated into control programs. In this study, we evaluated the diagnostic performance of urine metabolite biomarkers using a chemical reagent strip in the detection of S. haematobium infection in pre-school aged children. METHODS: A case-control study was conducted involving 82 pre-school aged children that were age and sex matched. Urine samples were collected for 3 consecutive days and were evaluated using urine filtration gold techniques as the gold standard method. The samples were simultaneously measured for metabolite biomarkers specifically haematuria, proteins, ketones, nitrites, glucose, bilirubin and urobilinogen using chemical reagent strips. Pearson correlation test was used to measure the relationship between S. haematobium infection and the urine metabolite biomarkers. RESULTS: The diagnostic performance of urine biomarkers were correlated with the microscopic examination urine filtration technique. Haematuria (r = 0.592, p = 0.0001) and proteinuria (r = 0.448, p = 0.0001) were correlated to S. haematobium infection. Negative correlations with p > 0.05 were recorded for ketones and urobilinogen. Highest sensitivity was 65.9 % (CI, 49.4 - 79.9) for haematuria whilst protein (albumin) biomarker had a lower specificity value of 43.9 % (28.5 - 60.3). Inversely, highest sensitivity was 87.8 % (73.8 - 95.9) for proteinuria whilst haematuria had a lower sensitivity value of 82.9 % (67.9 - 92.8). The positive predictive values ranged from 57.7 % (41.6 - 72.2) to 79.4 % (65.5 - 88.7) whereas negative predictive values ranged from 70.8 % (60.8 - 79.2) to 52.0 % (48.7 - 55.3). With respect to diagnostic efficiency, haematuria had a fair diagnostic performance with an area under the curve of 0.76 followed by proteinuria with proteinuria whilst the remaining metabolites fail discriminating ability with an area under the curve of <0.5. CONCLUSION: Although haematuria and protein biomarkers in urine are moderately sensitive and specific, they are important morbidity indicators of urogenital schistosomiasis in pre-school aged that may be utilised during screening in schistosomiasis control programs. We recommend comprehensive analysis of biomarkers using metabolomics techniques to identify novel urine biomarkers.

A high urinary urobilinogen/serum total bilirubin ratio indicates acute hepatic porphyria in patients with abdominal pain.

Acute hepatic porphyria (AHP) has always been a diagnostic dilemma for physicians due to its variable symptoms. Correct diagnosis mainly depends on the detection of an elevated urinary porphobilinogen (PBG), which is not a routine test and highly relies on the physician's awareness of AHP. In the present study, we identified a more convenient indicator during routine examinations to improve the diagnosis of AHP. We found that AHP patients showed a significant higher "FALSE" urinary urobilinogen level caused by urinary PBG during the urinalysis when detected by strips impregnated with Ehrlich reagent (P < 0.05). And a remarkable increase in the urinary urobilinogen/serum total bilirubin ratio was observed in AHP patients. The area under the ROC curve of this ratio for AHP was 1.000 (95% confidence interval 1.000-1.000, P < 0.01). A cutoff value of 3.22 for this ratio yielded a sensitivity of 100% and a specificity of 100% to distinguish AHP patients from the controls. Thus, we proved that a "falsely" high urinary urobilinogen level that was adjusted by the serum total bilirubin level (urinary urobilinogen/serum total bilirubin ratio) could be used as a sensitive and specific screening marker for AHP in patients with abdominal pain.

Annotation of the human adult urinary metabolome and metabolite identification using ultra high performance liquid chromatography coupled to a linear quadrupole ion trap-Orbitrap mass spectrometer.

Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)-based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.

Evaluation of the iChem® Velocity™ urine chemistry analyzer in a hospital routine laboratory.

BACKGROUND: The novel urine chemistry analyzer iChem Velocity (IRIS Diagnostics) offers improved urinalysis automation options through integration with the well-established iQ200 urine microscopy analyzer. In the course of optimizing the workflow in our hospital routine laboratory, we evaluated the performance of the iChem Velocity. METHODS: A total of 257 random urine samples were analyzed with the iChem Velocity, iQ200, Clinitek Atlas (Siemens Healthcare Diagnostics) and by manual microscopy. RESULTS: Depending on the parameter, 93% (hemoglobin) to 100% (urobilinogen), the iChem Velocity and Clinitek Atlas results agreed within the same rank or within one level of difference. The Clinitek Atlas featured a higher sensitivity for hemoglobin (area under the curve 0.86) and leukocyte esterase (area under the curve 0.85) compared with the iChem Velocity (area under the curve for hemoglobin 0.73, leukocytes 0.78). Imprecision was highest for hemoglobin and leukocytes in a pathological sample pool. While the precision of the Clinitek Atlas for hemoglobin measurements was superior, the iChem Velocity was more precise in analyzing protein and pH. CONCLUSIONS: Through urinalysis automation with the iChem Velocity and iQ200, we achieved a reduction of hands-on time by 89%. The sensitivity of this new system should be further improved through ongoing development.

Acute generalized exanthematous pustulosis and Coombs-positive hemolytic anemia in a child following Loxosceles reclusa envenomation.

Previously reported cases of acute generalized exanthematous pustulosis secondary to brown recluse spider bite have been questioned due to lack of identification of the spider or because of the concomitant administration of antibiotics. We report a 9-year-old boy who arrived at the emergency department with a confirmed Loxosceles reclusa bite to the neck. On the third day of hospitalization, he developed hundreds of monomorphous, sterile pustules, initially in intertriginous areas. The eruption disseminated and was followed by pinpoint desquamation typical for acute generalized exanthematous pustulosis. During this he also developed late onset Coombs-positive hemolytic anemia and systemic loxoscelism. Sphingomyelinase in Loxosceles venom induces the production of interleukin-8 and granulocyte-macrophage colony-stimulating factor, cytokines involved in the pathogenesis of acute generalized exanthematous pustulosis, providing a mechanism by which Loxosceles reclusa bite may trigger acute generalized exanthematous pustulosis. We suggest that this case adds Loxosceles envenomation to the spectrum of agents that can trigger acute generalized exanthematous pustulosis.

Association of UGT1A1 Gly71Arg with urine urobilinogen.

Bilirubin is glucoronized by uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1) mainly in the liver, and excreted into bile. The conjugated form is metabolized into the unconjugated form, and then into urobilinogen by bacteria in the intestine. Unconjugated bilirubin and urobilinogen are absorbed into the blood stream. The kidney filtrates conjugated bilurubin and urobilinogen into urine. Accordingly, the reduced enzyme activity of UGTIAI may decrease serum conjugated bilirubin levels, resulting in a lower frequency of positive results of urine bilirubin and urobilinogen. This study examined the associations of UGTIAI Gly71Arg (UGTIAI *6) with urine bilirubin and urobilinogen, as well as serum AST, ALT and GGT. Subjects were 5,172 inhabitants 35 to 69 years old who participated in a cohort study in Nagoya from June 2008 to May 2010. Among them, data from 5,151 participants (1,465 males and 3,686 females) were available for analysis. The age-sex-adjusted odds ratio (OR) of ArgArg relative to GlyGly was 1.37 (95% confidence interval (95% CI), 0.55-1.23) for bilirubin, and 1.67 (95% CI, 0.86-3.26) for urobilinogen. Those of ArgArg+ArgGly were 0.87 (95% CI, 0.59-1.27) and 1.50 (95% CI, 1.17-1.94), respectively. AST, ALT and GGT levels had no associations with the genotype. Although the significant association for urobilinogen was contrary to the biological expectation, this study indicated that UGTIA1 Gly71Arg may be a genetic factor of urine urobilinogen.

Abnormal findings on dipstick urinalysis of out-patients with malaria in Abakaliki, Nigeria.

BACKGROUND & OBJECTIVES: Malaria, one of the major health challenges of the tropics affecting about 500 million people, particularly the children and pregnant women have been associated with changes in urine compositions. The present study was undertaken to document the urinary abnormalities in malaria patients based on malaria species and the level of malaria parasitaemia. METHODS: Febrile patients (n = 365) with positive Giemsa - stained blood films for malaria recruited from Outpatient Department of Ebonyi State University Teaching Hospital, Abakaliki participated in the study. Patients were classified into two categories (+ and ++) based on parasite density. Apparently healthy individuals (n = 81), without malaria parasite on both thick and thin films of comparable age and gender acted as control group. Urine sample (10 ml) was collected from each participant and analysed using standard laboratory methods and techniques. RESULTS: Seventy - four (20.3%) of the patients had Plasmodium falciparum malaria. Although all the urine parameters were higher in the malarial patients in comparison to the control, only bilirubinuria and urobilinogenuria were statistically significant (p <0.05). Also, bilirubinuria, urobilinogenuria, haematuria and proteinuria were significantly (p < 0.05) higher in P. falciparum infection than in infections with other malaria species, but only in P. falciparum infection, bilirubinuria and urobilinogenuria were significantly (p < 0.05) higher at higher parasitaemia. CONCLUSION: Even though positive blood film for malaria parasite remains the gold standard for the diagnosis of malaria, urinary abnormalities, such as bilirubinuria, urobilinogenuria, proteinuria and haematuria may aid in identifying patients with severe malaria parasitaemia, especially the falciparum malaria.

Urine markers of kidney disorders and their risk associations in HIV-infected patients attending Nyanza Provincial General Hospital in Kisumu, Western Kenya.

OBJECTIVE: To identify abnormal levels of urine metabolites and cells that serve as markers of existing kidney disorders in ambulatory HIV-infected patients. DESIGN: A cross sectional study. SETTING: Nyanza Provincial General Hospital's patient support centre. SUBJECTS: A total of 593 HIV infected patients were studied. INTERVENTION: Dipstick urinalysis test was used to screen mid stream urine to detect constituents with altered levels. RESULTS: Out of the 593 participants, the urine of 214 (36.1%) had abnormally altered levels of urine constituents, with more females afflicted than males [41.5% vs. 27.8%; OR 1.84 (1.28-2.63), chi2 = 11.08, p = 0.0009]. Urobilinogen was the most common urine metabolite while ketones were least commonly present. More participants had altered levels of leucocytes than erythrocytes in urine. Patients with pyuria were three times more likely to have elevated erythrocytes in their urine as well (chi2 = 34.37, p < 0.0001). Similarly, the risk of having proteinuria was three times higher in patients with pyuria (p < 0.0003, Fisher's test). Patients with erythrocytes in urine also had a threefold likelihood of having proteinuria (P < 0.0003, Fisher's test). Fewer ARV users had abnormal urine markers [15.7% vs 24.3% OR 0.62 (0.41-0.94), chi2 = 5.2, p < 0.05]. CONCLUSION: Metabolites and cellular markers of kidney disorders were prevalent in the urine of HIV patients especially females and those with pronounced immune depletion (CD4 counts equal to or below 500). ARVs use was associated with reduced manifestation of these markers.

Carboxyhemoglobin elevation after exposure to dichloromethane.

Inhalation of dichloromethane vapor in concentrations of 500 to 1000 parts per million for 1 to 2 hours promptly initiated the formation of significant quantities of carbon monoxide in human subjects. The evidence suggests that carbon monoxide may be a metabolite of dichloromethane and, that exposure to concentrations of dichloromethane below the industrial threshold limit values may result in the formation of carbon monoxide in amounts that exceed the allowable limits.

Renal excretion of urobilinogen in the dog.

The renal excretion of urobilinogen was studied in dogs by standard clearance techniques. The use of radiochemically pure tritiated mesobilirubinogen as a representative urobilinogen afforded much greater analytical precision than can be obtained with the usual colorimetric and fluorimetric techniques which are only semiquantitative. With constant plasma levels of urobilinogen, raising urinary pH from 5 to 8 increased urobilinogen excretion from about 30% to up to 200% of the filtered load. When urinary pH was kept constant, changes in blood pH had no effect on urobilinogen excretion. Increases in urinary flow had no effect on urobilinogen excretion when the urine was alkaline but increased excretion markedly during aciduria. Probenecid did not influence urobilinogen excretion by the kidney. It is concluded that urobilinogen is excreted by a three-component system of glomerular filtration, active secretion, and pH-dependent nonionic diffusion in the distal nephron. Urobilinogen is a weak acid, and this mode of excretion is similar to that of other weak, organic acids, such as salicylates. These results indicate that urinary pH and flow must be considered in the clinical interpretation of measurements of urinary urobilinogen.

Urinalysis.

Urine analysis is an essential component of patient assessment, which is used for screening, diagnosis and planning care. This article discusses specimen collection and reagent strip testing.

Some aspects of bile acid and urobilinogen excretion and fecal elimination in men given a rural Guatemalan diet and egg formulas with and without added oat bran.

Six healthy men were fed a formula diet with and without oat bran and a natural food diet typical of rural Guatemala. No significant difference in dye transit time was found between diets but the Guatemalan diet significantly decreased dye retention time and increased stool frequency. Serum cholesterol and triglyceride levels showed no significant differences among dietary treatments. Excretion of fecal bile acids significantly increased on the Guatemalan and oat bran diets, but fecal bile acid concentration was significantly lower only on the Guatemalan diet. Urinary urobilinogen excretion and fecal urobilinogen concentration were significantly lower with the Guatemalan diet.

Laboratory tests and diagnostic procedures in evaluation of liver disease.

Evaluation of liver disease can be a difficult and imposing problem for general internists and noninternists alike. Physicians are often faced with a confusing array of what are commonly referred to as "liver function tests"; indeed, with the advent of and commonplace use of automated serum testing batteries, these findings are increasingly frequent in asymptomatic persons. Abnormalities in liver function are occasionally discovered incidentally when the testing battery is performed without suggestion of liver disease. There are numerous examples of algorithms and flow diagrams designed with an aim toward aiding clinicians in completion of an adequate diagnostic evaluation when faced with a particular set of abnormalities on "liver function tests." However, a clearer understanding of these tests and others, which are in a broader sense tests of liver function, might be of greater value than such a systematic and regimented approach to the evaluation of liver disease.

Urinary nitrite and urinary-tract infection.

Two dipstick procedures and an automated quantitative urinary nitrite assay were used to study nitrite in 786 samples of urine submitted to the bacteriology laboratory for routine culture and sensitivity testing. Many samples that had more than 100,000 nitrite-reducing organisms/ml and no detectable nitrite were studied. Limited nitrate concentration in urine was not a significant cause of false-negative nitrite results. However, in some urine samples nitrite added in vitro was lost during a four-hour incubation in vitro at 37 C in the presence of more than 100,000 nitrite-reducing organisms/ml. Ascorbic acid, abnormal amounts of urobilinogen, and urinary pH below 6.0 are all possible causes of false-negative nitrite determinations.

Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex.

Previous methods to quantitate urobilinogen lack precision due to either incomplete reduction of urobilin or to losses of pigment before the use of Ehrlich's aldehyde reaction or due to pigment precipitation, as occurs in Schlesinger's fluorescent assay. The present procedure modifies the latter assay to obviate described problems as it is based on direct spectrophotometry (or spectrofluorometry) of a zinc complex of urobilin in dimethylsulfoxide. The sample is extracted with dimethylsulfoxide to increase recovery of urobilinogen from samples of various origin (feces, urine, bile, serum etc.) and to prevent the precipitation of proteins. After oxidation of urobilinogen with iodine, the concentration of the resulting urobilin is directly determined from the absorption (or fluorescent) spectrum. High sensitivity and high specificity for the procedure result from the high value of absorption coefficient and by the characteristic absorption spectrum of zinc complex of urobilin, respectively. Within-day and day-to-day coefficients of variation of stool and bile samples range from 1.6 to 9.2%. The smallest concentration of urobilinogen measurable by spectrophotometry is approximately 0.5 mumol/l, by fluorometry it is 0.25 mumol/l. The recovery varies from 82.2 to 93.8% depending on re-extraction of the sample. The method is linear in the range of 1 to 35 mumol/l and of 0.5 to 17.5 mumol/l for spectrophotometric and fluorescent determinations, respectively. The results obtained with the present method correlated well with Ehrlich's determination (r2 = 0.912), but are approximately two-fold higher. Storage of the samples at -20 degrees C or extraction with dimethylsulfoxide prior to storage are good ways for sample preservation. Twenty stool samples from healthy adults were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinalysis: its proper role in the physician's office.

Urine study is an important aspect of physician's office laboratories. The contributions that urine study can make are important for both the patient and the physician. State-of-the-art technology makes it possible to have urine test results available at the time the patient is being examined without inconvenience to the patient, the physician, or the laboratory. The cost-containment contributions of urine study are significant for both the patient and the physician. The analytes currently studied provide a broad spectrum of information that relates to many of the problems presented by the typical patient in the typical physician's office.

Biochemical monitoring of the surgical patient.

The need for prognostic and biochemical monitors of cellular function becomes increasingly evident with the further elucidation of the metabolic pattern secondary to disease. Thus far studies have shown that lactic acid may be one of these biochemical parameters. Investigations are now in the process of defining other biochemical indicators of cellular dysfunction.