Complex In Urine Research Articles

A novel method for rapid and quantitative detection of bisphenol A in urine.

Bisphenol A (BPA) is classified as an endocrine disruptor (ED) and it can interact with variety of hormone receptors leading to hormonal disruption and increased risk of various adverse health effects. Reducing human exposure to BPA is one of the main challenges of public health, as it is constantly present in daily life. A low-cost and commonly applied method to enable determination of BPA in the patient's body has yet to be developed. Currently available techniques are expensive, time-consuming, and require access to highly equipped analytical chemistry laboratories. Here we describe a fast and cheap engineered lateral flow assay of our design, to detect of BPA in urine samples. The technology not only provides an opportunity to perform rapid medical diagnostics without the need for an access to the central laboratory but also a means for self-diagnosis by the patient. The addition of β-glucuronidase improves the sensitivity of detection as it releases the free BPA from glucuronide complexes in urine. This invention may become a demonstrated analytical means for lowering human exposure to BPA and probably also to other EDs and consequently, may be useful in decrease of the risk for several lifestyle diseases.

Chemical equilibria of complexes in urine. A contribution to the physicochemistry of infectious urinary stone formation

The formation of infectious urinary stones is a complex physicochemical process that occurs in urine. Infectious urinary stones are formed only when the urinary tract is infected by microorganisms producing urease. The presence of urease-positive bacteria in urine causes the elevation of the concentration of ammonia and carbon dioxide. As a consequence, the presence of bacteria in the solution of urine causes the changes in concentration of various ions and influences on the chemical equilibria of various complexes in urine. In this paper, the analysis of chemical complexes created in the solution of artificial urine of basic composition and with additional quantity of ammonia and carbon dioxide is presented. These two cases correspond to the situation without and with infection within the urinary tract, respectively. In other words, in this paper we try to answer to the question how the presence of urease-positive bacteria in urine influences on the chemical equilibria of various complexes in urine.

Biomarker discovery in mass spectrometry-based urinary proteomics.

Urinary proteomics has become one of the most attractive topics in disease biomarker discovery. MS-based proteomic analysis has advanced continuously and emerged as a prominent tool in the field of clinical bioanalysis. However, only few protein biomarkers have made their way to validation and clinical practice. Biomarker discovery is challenged by many clinical and analytical factors including, but not limited to, the complexity of urine and the wide dynamic range of endogenous proteins in the sample. This article highlights promising technologies and strategies in the MS-based biomarker discovery process, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics. Different proteomics approaches are discussed, and progresses in maximizing urinary proteome coverage and standardization are emphasized in this review. MS-based urinary proteomics has great potential in the development of noninvasive diagnostic assays in the future, which will require collaborative efforts between analytical scientists, systems biologists, and clinicians.

Evaluation of MMP-2, MMP-9, TIMP-1, TIMP-2, NGAL and MMP-9/NGAL complex in urine and sera from patients with bladder cancer.

The identification of biomarkers in urine or serum samples from patients with bladder cancer is urgently required for the development of non-invasive methods for the diagnosis of bladder carcinoma and to facilitate follow-up surveillance, to combat the high progression and recurrence rates of this type of cancer. The current study measured the content of matrix metalloproteinase (MMP)-2 and -9, as well as tissue inhibitor of metalloproteinase (TIMP)-1 and -2 in the urine and sera of 41 patients with bladder cancer by ELISA. The association between levels of MMP-2 and -9 and TIMP-1 and -2, and tumor grade and stage were investigated to verify whether these molecules are involved in tumor differentiation. Statistical analysis of the data revealed that urinary TIMP-1 levels were significantly higher in the high grade group compared with those of the low grade samples (P=0.022). The results also revealed a significantly differing distribution of TIMP-1 expression between Ta and T1 stage specimens (P=0.040). The corresponding area under the curves (AUCs) were 0.72, with a sensitivity of 0.70 and specificity of 0.75. In addition, neutrophil gelatinase-associated lipocalin (NGAL) and MMP-9/NGAL complex levels in the sera were measured. All molecules evaluated were detected in the sera of the patients studied. In particular, tumors staged as non-muscle invasive (Ta and T1), demonstrated significantly higher NGAL levels compared with those of muscle invasive (>T1) bladder cancer (32.8 ng/ml vs. 16.2 ng/ml; P=0.029). The discriminatory ability of NGAL expression was confirmed by receiver operating characteristic curve analysis that revealed an AUC of 0.75, a sensitivity of 0.88 and a specificity of 0.67. These data indicated that urinary TIMP-1 and serum NGAL may be useful non-invasive biomarkers to provide clinical information for bladder cancer disease management. Multicenter, prospective studies are required to confirm these preliminary results.

Antigen‐specific immune complexes in urine of patients with lymphatic filariasis

Lymphatic filarial subjects with disease manifestations exhibit significantly elevated levels of immune complexes (ICs) in their circulation. The objective of the study was to explore the possible excretion of filaria-specific soluble ICs in urine of subjects with lymphatic filariasis. Paired urine (overnight) and serum samples were analyzed for complement activating filarial antigen containing immune complexes by enzyme-linked immunosorbent assay (ELISA). Antigen-specific ICs were detected in urine samples of 34% of subjects with filarial disease manifestations while the frequency of occurrence was low in microfilaremic subjects. The titer of urine ICs is significantly high in subjects with chronic filariasis as compared to microfilaria (mf) carriers. The occurrence of filaria-specific ICs in urine and their passage through the filtering structures of the kidney is suggestive of the focal or diffuse damage in those subjects. Detection of ICs in urine may provide a noninvasive means of assessing the extent of renal damage in patients with lymphatic filariasis.

Therapeutic action of citrate in urolithiasis explained by chemical speciation: increase in pH is the determinant factor

The therapeutic action of citrate in the management of calcium oxalate urolithiasis has been attributed to the depletion of free calcium ions by complexation of the latter by citrate itself. However, little attention has been given to the nature of such complexes and the chemical conditions which control their formation because it is very difficult to measure them in solution. We therefore modelled the theoretical formation of these complexes in urine following administration of a citrate-containing preparation, using a powerful speciation program, JESS (Joint Expert Speciation System), which has been widely used to model metal-ligand equilibria in biological systems but which has hitherto not been applied in urolithiasis research. This program has an extensive database of thermodynamic constants and is able to calculate mixed ligand speciation. Urine data obtained before and after citrate administration in four groups of subjects (male and female normals and stone formers) were used as input for JESS to calculate the speciation of calcium, citrate and oxalate. The program was also used to examine the effects of varying different urinary components on the nature and concentration of the various species. The speciation predicted the formation of a key calcium-citrate-phosphate species (previously unreported in urolithiasis research), which accounts for a significant percentage of the complexation of the free calcium. Moreover, the formation of this complex was found to be dependent on an increase in urinary pH rather than on an increase in urinary citrate concentration per se. The therapeutic action of citrate in the management of calcium oxalate urolithiasis is due to the formation of a pH dependent calcium-citrate-phosphate complex which reduces the concentration of the free calcium ion species, thereby reducing the risk of stone formation.

Effects of trace metals on the inhibition of calcium oxalate crystallization

The aim of this study was to examine the possible effects of some trace metals on the inhibition of calcium oxalate crystallization. A test of urinary lithogenic risk was used to follow the crystallization of calcium oxalate from artificial urine in the presence of several metal ions assayed in their physiological concentrations. Interactions of these metal ions with known inhibitors of such crystallization (phytate, pyrophosphate, citrate and chondroitin sulphate) were also investigated. None of the metals affected the inhibition of calcium oxalate crystallization at concentrations approximating those found in normal urine, with the exception of the Fe3+ ions. Interactions of Fe3+ with some urinary components produced both synergic (phytate and pyrophosphate) and negative (citrate) effects on preventing crystallization. These effects are explained in terms of the affinity of the inhibitors for the calcium oxalate crystal surface and their ability to form stable complexes in urine. Because of the minimal concentrations, we conclude that physiological concentrations of trace elements in urine have no significant influence on calcium oxalate crystallization. In this sense, ferric ions, which exhibit an intrinsic high inhibitory capacity of calcium oxalate crystallization at physiological concentrations, even increased by the concomitant presence of phytate and pyrophosphate, are probably unable to act as powerful inhibitors in the presence of physiological urinary concentrations of citrate, due to the formation of highly stable complexes in solution without inhibitory activity.

PCR-based detection of the Mycobacterium tuberculosis complex in urine of HIV-infected and uninfected pulmonary and extrapulmonary tuberculosis patients in Burkina Faso

To evaluate a one-tube nested PCR-based analysis of urine for diagnosing pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) in Bobo-Dioulasso, Burkina Faso, a prospective analysis of urine samples from HIV- and non-HIV-infected adults with PTB and EPTB (case patients) and with pathology other than tuberculosis (TB) (control patients) was performed. Three groups of patients were classified as microbiological-positive and -negative PTB and EPTB on the basis of clinical signs and microbiological results. Urine from patients was analysed using the DNA extraction and Sechi's methods, both modified, for the detection of Mycobacterium tuberculosis. The sensitivity, specificity, positive predictive value and negative predictive value were calculated. The sensitivity of the test for the microbiological-positive PTB, microbiological-negative PTB and EPTB was 40.5 % (88/217), 66.7 % (20/30) and 57.1 % (48/84), respectively. The specificity was 98.2 %. Differences were observed in the two populations infected and not infected by HIV. This method is not appropriate for detection of new TB cases in the routine laboratory, but it can be useful for cases where the clinical and bacteriological diagnosis of TB is not conclusive.

Determination of chromium in urine samples by complexation–supercritical fluid extraction and liquid or gas chromatography

A method was developed for the determination of chromium as the Cr(acac) 3 complex in urine using SFE and chromatography. Quantitative extractions were achieved when the experiments were carried out under 3000 p.s.i. of pressure, at a temperature of 120 °C, with 2.0 ml of methanol, 30 min of static extraction and 5 min of dynamic extraction. The chromium was quantified by GC–FID detection. The calibration graph of Cr(acac) 3 solutions was linear between 0.50 and 43.0 μg ml −1 of chromium (DL 0.18 μg ml −1, R=0.9994). The same extracts were quantified by HPLC-340 nm detection. The calibration curve of the Cr(acac) 3 solutions was linear over a range of 0.013 to 60.0 μg ml −1 (DL 0.02 μg ml −1, R=0.9999). This method was applied to urine samples from 60 diabetic patients and 21 healthy volunteers. Chromium concentration ranges were 2.5–29.5 μg l −1 for the diabetics and 5.9–12.3 μg l −1 for the normal subjects.

Detection of Na(+) transporter proteins in urine.

Previous studies have established that the vasopressin-regulated water channel of the collecting duct, aquaporin-2, is excreted in the urine, providing a means for assessment of regulation and dysregulation of aquaporin-2 in humans. This article addresses the hypothesis that membrane transporters from upstream nephron segments are normally detectable in urine. The experiments employed rabbit polyclonal antibodies against the major Na transporters of the proximal tubule (the type 3 Na-H exchanger [NHE3]), the thick ascending limb of Henle's loop (the bumetanide-sensitive Na-K-2Cl cotransporter [NKCC2]), and the distal convoluted tubule (the thiazide-sensitive Na-Cl cotransporter [NCC]) in immunoblotting experiments. All three of these transporters were readily detectable as high molecular weight complexes present in lowdensity membrane fractions from urine of normal rats. Cross linking studies of NHE3, NKCC2, and NCC revealed that high molecular weight complexes are normally present in renal tissue. The molecular weights of the complexes in urine matched those of the cross-linked complexes in native kidney tissue. The presence in urine of integral membrane proteins representative of each nephron segment raises the possibility that limited or comprehensive proteomic analysis of urine samples may be useful in clinical settings.

Oxygen radical mediated modification of a proteins and neurodegenerative disorders in aged patients

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (·NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-d-glucamine dithiocarbamate-ferrous iron, [(MGD)2/Fe], which binds to ·NO and forms a water-soluble [(MGD)2/Fe-NO] complex. At 2 h after injection of the [(MGD)2/Fe] complex, a three-line EPR signal characteristic of the [(MGD)2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 μM respectively. This 25-fold increase in ·NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of ·NO in mice. Administration of N-monomethyl-l-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15n-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14 NO] complex and a two-line spectrum of the [(MGD)2/Fe-15 NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected ·NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible ·NO synthase activities in living animals under physiological as well as pathophyiological conditions where ·NO is overproduced.

THE BLADDER TUMOR ANTIGEN (BTA) TEST COMPARED TO VOIDED URINE CYTOLOGY IN THE DETECTION OF BLADDER NEOPLASMS

Purpose Tests to detect recurrent bladder neoplasms are limited and none is consistently accurate. Recent studies suggest that the bladder tumor antigen (BTA * * C.R. Bard, Inc., Redmond, Washington. ) test, an agglutination reaction for basement membrane complexes, is superior to voided urine cytology in clinical practice. We compared BTA and voided urine cytology to bladder washings and cystoscopy, emphasizing diagnostic yield among patients with causes of basement membrane complexes other than bladder cancer. Materials and Methods Random voided urine specimens from 67 patients with a history of bladder neoplasms were collected before cystoscopy and bladder washing. Urine also was obtained from 34 patients with inflammatory bladder conditions including 5 with a history of prostate cancer. Each urine was tested for BTA according to a commericial kit. Positive results were indicated by yellow on a test pad. Blinded to all other results, each urine and each bladder washing were examined microscopically, and a positive test had malignant/suspicious cells. Bladder biopsies were performed when endoscopic lesions were seen. Specimens were grouped into 4 categories: group 1-biopsy proved bladder neoplasm, group 2-history of bladder cancer but not biopsy proved, group 3-history of prostate cancer and group 4-no history of urological cancer. Results Voided urine cytology was positive in 54% of specimens from patients with biopsy proved bladder neoplasms compared to 29% for BTA. Relative yield for voided urine cytology versus BTA was not changed if all group 2 cases having a positive bladder washing and positive cystoscopy were assumed to have bladder cancer, nor was relative yield altered by subsequent short-term followup. Of voided urine specimens 14% from group 1 patients and 41% from group 2 patients had scant cells. Overall diagnostic yield was superior for bladder washing. False-positive BTA occurred in 7 of 34 patients with no history of urological or prostate cancer. There were no false-positive voided urine cytology interpretations in these groups. Conclusions BTA is not superior to voided urine cytology in detecting bladder neoplasms and may be limited by false-positive reactions in patients with other causes of basement membrane complexes in urine. Voided urine samples may be limited by high frequency of hypocellularity. Of 34 patients with a hypocellular urine specimen 4 had biopsy proved bladder cancer. Bladder washing yields best results but requires instrumentation. No test, including cystoscopy, is accurate always.

Polymerase Chain Reaction for Detection of Myocobacterium Tuberculosis in Clinical Specimens

In order to determine the senstivity of the polymerase chain raaction (PCR) for rapid diagnosis of tuberculosis, 157 clinical specimens of sputum (n=111), urine (n=29), cerebrospinal fluid (n=6) and others (n=11) were examined with PCR and conventional method. Of these samples, 68 were obtained from tuberculosis patients (33 from smear and/or culture positive for Mycobacterium tuberculosis; 35 from patients with clinically active, smear and/ or culture negative), 29 from patients whose antituberculous treatment was interrupted one year previously, 59 from patients without any definitive diagnosis for tuberculosis and one from a patient whose sputum yielded atypcial mycobacterium. The PCR positivity for these groups was 76.4%, 24.1% and 25.4%, respectively. The polymerase chain reaction was negative in the sample from which atypical mycobacterium was isolated. The sensitivity of the PCR was found to be 100 % for the specimens with smear and/or culture positive for Mycobacterium tuberculosis complex (33/33). PCR was found positive in 39 (81.2%) of 48 sputum, 7 (87.5%) of 8 urine, 2 (67%) of 3 cerebrospinal fluid and 4 (44.4%) of 9 other specimens from active tuberculosis patients. These results indicate that the polymerase chain reaction can be used to detect Mycobacterium tuberculosis complex in urine and cerebrospinal fluid as reliably as in sputum.

Complexes of tissue kallikrein with protein C inhibitor in human semen and urine.

An ELISA was developed for quantifying the complex between tissue kallikrein (tKK) and protein C inhibitor (PCI) (tKK:PCI) in seminal plasma and urine. The ELISA used purified tKK:PCI complex as a standard and was specific for this complex with a detection limit of about 1.1 pM. Purified tKK:PCI complex was obtained from human urine and was 95% homogeneous as judged by SDS/PAGE. The 90-kDa band corresponding to the purified tKK:PCI complex reacted with anti-tKK and anti-PCI antibodies as judged by immunoblotting. Seminal plasma collected in the absence of extrinsic inhibitors contained 1.8 +/- 0.6 nM tKK:PCI complex and 4.7 +/- 2.8 nM immunoreactive tKK (mean +/- SD, n = 10), which indicates that about 28% of the total tKK immunoreactivity is forming complexes with PCI. When semen was collected in the presence of tKK inhibitors it had only about 6% of the tKK complexed to PCI. In vitro studies showed that the tKK:PCI complex formation in semen was accomplished in about 1 h and that heparin stimulated both the rate and the extent of complexation of tKK with PCI. Native urine showed low levels of tKK:PCI complex, but after dialysis urine had 0.17 +/- 0.05 nM complex. Formation of tKK:PCI complex in urine and semen was also demonstrated by immunoblotting. These results suggest that PCI is a physiological inhibitor of tKK and provide additional evidence of the involvement of PCI in human reproduction.

Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (·NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N- methyl- d - glucamine dithiocarbamate-ferrous iron, [(MGD) 2/Fe], which binds to ·NO and forms a water-soluble [(MGD) 2/Fe-NO] complex. At 2 h after injection of the [(MGD) 2/Fe] complex, a three-line EPR signal characteristic of the [(MGD) 2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD) 2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 μM respectively. This 25-fold increase in ·NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of ·NO in mice. Administration of N-monomethyl- l-arginine ( NMMA; 50 mg/ kg) inhibited the ex vivo signal intensities of the [(MGD) 2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15 n-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD) 2/Fe- 14 NO] complex and a two-line spectrum of the [(MGD) 2/Fe- 15 NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected ·NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible ·NO synthase activities in living animals under physiological as well as pathophyiological conditions where ·NO is overproduced.

Pt(CBDCA-O)(NH3)2(L-Methionine-S)]: Ring-Opened Adduct of the Anticancer Drug Carboplatin ("Paraplatin"). Detection of a Similar Complex in Urine by NMR Spectroscopy

Pt(CBDCA-O)(NH3)2(L-Methionine-S)]: Ring-Opened Adduct of the Anticancer Drug Carboplatin ("Paraplatin"). Detection of a Similar Complex in Urine by NMR Spectroscopy

Immunological properties of fibrin high affinity urokinase inhibitor complex in urine and plasma

The immunological properties of fibrin high affinity urokinase inhibitor complex, a urokinase and PAI (plasminogen activator inhibitor) were measured. The fibrin high affinity urokinase inhibitor complex formed a precipitating arc against antifibrin high affinity urokinase inhibitor complex serum during immunoelectrophoresis. Urokinase-containing plasma also formed an arc in the at region against the same antiserum. Immunoblotting of fibrin high affinity urokinase inhibitor complex demonstrated a band of Mr 100 kDa against antifibrin high affinity urokinase inhibitor complex antibody but failed to demonstrate the same band againt anti-PAI-1 antibody. Urine which had passed through fibrin-Sepharose and urine which was eluted from fibrin-Sepharose both formed bands at Mr 100 kDa, suggesting that urine contains two types of urokinase (two chain) and PAT complexes, which may or may not show fibrin affinity, and that the complex which does show fibrin affinity can be converted to the complex which does not. In plasma, a urokinase and PAI complex (Mr 100 kDa) which was immunologically identical to fibrin high affinity urokinase inhibitor complex was detected by immunoblotting. When urokinase infused into thrombotic patients, such a complex either showed slightly increased intensity or a complex of Mr 200 kDa appeared.

128 Immunological properties of urokinase-urokinase inhibitor complex in urine and plasma

128 Immunological properties of urokinase-urokinase inhibitor complex in urine and plasma

Immune Complexes in Urine and Serum of Patients with Bladder Cancer

The urine and/or serum of 43 patients with bladder cancer was tested for the presence of immune complexes. Immune complexes were present more frequently in the urine (38 per cent) than in the serum (23 per cent). Urinary immune complexes were elevated in 10 per cent of the patients with no tumor (group 1), 25 per cent with superficial neoplasms (group 2) and 89 per cent with infiltrating or metastatic disease (group 3), which compared favorably to elevated serum immune complexes in 20, 25 and 33 per cent of the patients, respectively. The incidence of urinary immune complexes was statistically significant when compared in patients with and without active tumors (p less than 03), and when group 3 patients were compared to those in group 1 or 2 (p less than 0.01 and p less than 0.03, respectively). Unlike serum immune complexes urinary immune complexes appeared to correlate with the presence and stage of bladder cancer

URINARY PREGNANEDIOL DETERMINATION AS A TEST OF PREGNANCY*

IN 1936 Venning and Browne (1) described the isolation of a water-soluble pregnanediol complex from human pregnancy urine. In 1937 Venning, Henry and Browne (2) described the measurement of this complex and the same year Venning (3) published a gravimetric method for determination of sodium pregnanediol glucuronidate in urine, and presented evidence of its metabolism from progesterone. Venning and Browne (4) and Venning, Henry and Browne (5), using this procedure, reported the presence of significant amounts of the complex in urine from women in the luteal phase of the menstrual cycle. Subsequently, these workers and many others using the same procedure, investigated the urinary excretion of sodium pregnanediol glucuronidate in pregnant and nonpregnant women (6–18). Urinary sodium pregnanediol glucuronidate levels were reported to be of value in the diagnosis of pregnancy, of threatened abortion, and determination of the date of ovulation, all states reflecting a change in luteal activity. Since Venning's...