ABSTRACT
Previous research suggests that the neuropeptide orexin A contributes to sympathetic blood pressure (BP) control inasmuch as hypothalamic injection of orexin A increases sympathetic vasomotor tone and arterial BP in rodents. In humans with narcolepsy, a disorder associated with loss of orexin-producing neurons, vasoconstrictive muscle sympathetic nerve activity (MSNA) is reduced. Since intranasally administered oligopeptides like orexin are known to modulate brain function, we investigated the effect of intranasal orexin A on vascular sympathetic baroreflex function in healthy humans. In a balanced, double-blind crossover study, orexin A (500 nmol) and placebo, respectively, were intranasally administered to 10 lean healthy males (age 25.8 ± 4.6 yr). MSNA was assessed microneurographically before and 30-45 min after either substance administration. Additionally, baroreflex was challenged via graded infusions of vasoactive drugs before and after substance administration. Baroreflex function was defined as the correlation of BP with MSNA and heart rate. Intranasal orexin A compared with placebo induced a significant increase in resting MSNA from pre-to postadministration [Δburst rate, orexin A vs. placebo: +5.8 ± 0.8 vs. +2.1 ± 0.6 bursts/min, P = 0.007; total activity 169 ± 11.5% vs. 115 ± 5.0%; P = 0.002]. BP, heart rate, and sympathovagal balance to the heart, as represented by heart rate variability (HRV), as well as baroreflex sensitivity during the vasoactive challenge were not altered. Intranasally administered orexin A acutely induced vasoconstrictory sympathoactivation in healthy male humans. This result suggests that orexin A mediates upward resetting of the vascular baroreflex set point at centers superordinate to the mere baroreflex feedback loop.NEW & NOTEWORTHY Our pilot study adds another important part to the complex network of neuroendocrine-sympathetic interaction. Our results demonstrate that intranasal orexin A elicits an excitatory effect on sympathetic vascular tone superordinate to mere baroreflex feedback regulation. This resetting of the baroreflex set point suggests an activation of hypothalamic core centers such as the paraventricular nucleus (PVN). The role of the orexinergic system in the development of neurogenic arterial hypertension warrants further investigations.
Subject(s)
Baroreflex/drug effects , Blood Pressure/drug effects , Heart Rate/drug effects , Orexins/pharmacology , Sympathetic Nervous System/drug effects , Vasoconstriction/drug effects , Administration, Intranasal , Adult , Cross-Over Studies , Double-Blind Method , Humans , Male , Orexins/administration & dosage , Pilot Projects , Young AdultABSTRACT
Oxytocin appears to be involved in the neuroendocrine regulation of sympathetic blood pressure (BP) homeostasis. In animals, intracerebral administration of oxytocin induces BP-relevant sympathetic activation. In humans, central nervous effects of oxytocin on BP regulation remain unclear. Intranasal administration supposedly delivers oligopeptides such as oxytocin directly to the brain. We investigated the effects of intranasal oxytocin on sympathetic vascular baroreflex function in humans using microneurographic techniques. In a balanced, double-blind crossover design, oxytocin or placebo was administered intranasally to 12 lean, healthy males (age 25 ± 4 yr). Muscle sympathetic nerve activity (MSNA) was assessed microneurographically before (presubstance), 30-45 min (postsubstance I), and 105-120 min (postsubstance II) after oxytocin administration. Baroreflex was challenged via graded infusions of vasoactive drugs, and correlation of BP with MSNA and heart rate (HR) defined baroreflex function. Experiments were conducted in the afternoon after a 5-h fasting period. After oxytocin, resting MSNA (burst rate and total activity) showed significant net increases from pre to postsubstance II compared with placebo [Δincrease = +4.3 ± 1.2 (oxytocin) vs. +2.2 ± 1.4 bursts/min (placebo), ANOVA; P < 0.05; total activity = 184 ± 11.5% (oxytocin) vs. 121 ± 14.3% (placebo), ANOVA; P = 0.01). This was combined with a small but significant net increase in resting diastolic BP, whereas systolic and mean arterial BP or HR as well as baroreflex sensitivity at vasoactive drug challenge were not altered. Intranasally administered oxytocin induced vasoconstrictory sympathoactivation in healthy male humans. The concomitant increase of diastolic BP was most likely attributable to increased vascular tone. This suggests oxytocin-mediated upward resetting of the vascular baroreflex set point at centers superordinate to the mere baroreflex-feedback loop.
Subject(s)
Oxytocics/administration & dosage , Oxytocics/pharmacology , Oxytocin/administration & dosage , Oxytocin/pharmacology , Administration, Intranasal , Adult , Cross-Over Studies , Double-Blind Method , Humans , Male , Nitroglycerin/administration & dosage , Nitroglycerin/pharmacology , Phenylephrine/administration & dosage , Phenylephrine/pharmacology , Sympathomimetics/administration & dosage , Sympathomimetics/pharmacology , Vasodilator Agents/administration & dosage , Vasodilator Agents/pharmacology , Young AdultABSTRACT
Expression of proximal tubular organic anion transporters Oat1 and Oat3 is reduced by PGE2 after renal ischemia and reperfusion (I/R) injury. We hypothesized that impaired expression of Oat1/3 is decisively involved in the deterioration of renal function after I/R injury. Therefore, we administered probenecid, which blocks proximal tubular indomethacin uptake, to abolish the indomethacin-mediated restoration of Oat1/3 regulation and its effect on renal functional and morphological outcome. Ischemic acute kidney injury (iAKI) was induced in rats by bilateral clamping of renal arteries for 45 min with 24-h follow-up. Low-dose indomethacin (1 mg/kg) was given intraperitoneally (ip) at the end of ischemia. Probenecid (50 mg/kg) was administered ip 20 min later. Indomethacin restored the expression of Oat1/3, PAH net secretion, and PGE2 clearance. Additionally, indomethacin improved kidney function as measured by glomerular filtration rate (GFR), renal perfusion as determined by corrected PAH clearance, and morphology, whereas it reduced renal cortical apoptosis and nitric oxide production. Notably, indomethacin did not affect inflammation parameters in the kidneys (e.g., monocyte chemoattractant protein-1, ED1+ cells). On the other hand, probenecid blocked the indomethacin-induced restoration of Oat1/3 and moreover abrogated all beneficial effects. Our study indicates that the beneficial effect of low-dose indomethacin in iAKI is not due to its anti-inflammatory potency, but in contrast to its restoration of Oat1/3 expression and/or general renal function. Inhibition of proximal tubular indomethacin uptake abrogates the beneficial effect of indomethacin by resetting the PGE2-mediated Oat1/3 impairment, thus reestablishing renal damage. This provides evidence for a mechanistic effect of Oat1/3 in a new model of the induction of renal damage after iAKI.
Subject(s)
Acute Kidney Injury/metabolism , Ischemia/drug therapy , Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Reperfusion Injury/drug therapy , Acute Kidney Injury/drug therapy , Acute Kidney Injury/physiopathology , Animals , Disease Models, Animal , Female , Glomerular Filtration Rate/drug effects , Indomethacin/administration & dosage , Indomethacin/pharmacology , Ischemia/metabolism , Kidney/metabolism , Rats, Sprague-Dawley , Reperfusion Injury/metabolismABSTRACT
Renal organic cation transporters are downregulated by nitric oxide (NO) in rat endotoxemia. NO generated by inducible NO synthase (iNOS) is substantially increased in the renal cortex after renal ischemia-reperfusion (I/R) injury. Therefore, we investigated the effects of iNOS-specific NO inhibition on the expression of the organic cation transporters rOct1 and rOct2 (Slc22a1 and Slc22a2, respectively) after I/R injury both in vivo and in vitro. In vivo, N(6)-(1-iminoethyl)-L-lysine (L-NIL) completely inhibited NO generation after I/R injury. Moreover, L-NIL abolished the ischemia-induced downregulation of rOct1 and rOct2 as determined by qPCR and Western blotting. Functional evidence was obtained by measuring the fractional excretion (FE) of the endogenous organic cation serotonin. Concordant with the expression of the rate-limiting organic cation transporter, the FE of serotonin decreased after I/R injury and was totally abolished by L-NIL. In vitro, ischemia downregulated both rOct1 and rOct2, which were also abolished by L-NIL; the same was true for the uptake of the organic cation MPP. We showed that renal I/R injury downregulates rOct1 and rOct2, which is most probably mediated via NO. In principle, this may be an autocrine effect of proximal tubular epithelial cells. We conclude that rOct1, or rOct1 and rOct2 limit the rate of the renal excretion of serotonin.
Subject(s)
Cation Transport Proteins/metabolism , Kidney Diseases/metabolism , Kidney/metabolism , Nitric Oxide/pharmacology , Reperfusion Injury/metabolism , 1-Methyl-4-phenylpyridinium/metabolism , Animals , Blotting, Western , Catecholamine Plasma Membrane Transport Proteins/biosynthesis , Cell Line , Down-Regulation , Enzyme Inhibitors/pharmacology , Female , Kidney/drug effects , Kidney Cortex/drug effects , Kidney Cortex/metabolism , Kidney Function Tests , Lysine/analogs & derivatives , Lysine/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Organic Anion Transporters, Sodium-Independent/biosynthesis , Organic Cation Transport Proteins/biosynthesis , Organic Cation Transporter 2 , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Serotonin/urineABSTRACT
We have previously shown that expression of renal organic anion transporters Oat1 and Oat3 is diminished by prostaglandin E(2) (PGE(2)) and that both transporters are downregulated after renal ischemia. Because PGE(2) is increased after renal ischemia and is generated by cyclooxygenases (COX), we investigated the effect of the COX inhibitor indomethacin on expression of Oat1/3 after ischemic acute kidney injury (iAKI). iAKI was induced in rats by bilateral clamping of renal arteries for 45 min. Indomethacin (1 mg/kg) was given intraperitoneally as soon as reperfusion started. Sham-treated animals served as controls. Oat1/3 were determined by qPCR and Western blot. PGE(2) in blood and urine was measured by enzyme-linked immunosorbent assay. Invasion of monocytes/macrophages was determined. Glomerular filtration rate and renal plasma flow were determined. All parameters were detected 24 h after ischemia. PAH net secretion, as well as clearance and secretion of PGE(2) were calculated. In clamped animals, indomethacin restored expression of Oat1/3, as well as PAH net secretion, PGE(2) clearance, or PGE(2) secretion. Additionally, indomethacin substantially improved kidney function as measured by glomerular filtration and PAH clearance. Indomethacin did not affect ischemia-induced invasion of monocytes/macrophages. In conclusion, our study indicates that low-dose indomethacin applied after ischemia prevents ischemia-induced downregulation of Oat1/3 during reperfusion and has a substantial protective effect on kidney function after iAKI. The beneficial effect of low-dose indomethacin on renal outcome is likely due to an effect different from inhibition of inflammation. In accordance to the decreased PAH net secretion, renal excretion of an endogenous organic anion (PGE(2)) is also impaired after ischemia and reperfusion.
Subject(s)
Cyclooxygenase Inhibitors/administration & dosage , Indomethacin/administration & dosage , Kidney/blood supply , Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Reperfusion Injury/metabolism , Acute Disease , Animals , Biological Transport/drug effects , Dinoprostone/metabolism , Dinoprostone/urine , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Female , Kidney/metabolism , Kidney/physiopathology , Kidney Cortex/pathology , Monocytes/pathology , Rats , Rats, Sprague-Dawley , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , p-Aminohippuric Acid/metabolismABSTRACT
Modifications of the Zwikker- and Parri color detection tests were investigated and compared according to their ability to distinguish between nine different barbituric acids and hydantoins. Solutions of the resulting complexes in 50% DMSO were analyzed spectrophotometrically. 350 spectra have been analyzed and criteria for their assessment have been defined. The evaluation based upon the occurrence of a peak in the visible absorption spectra, in comparison with the spectrum of the blank solution. The results were in accordance to those obtained in the visual assessment using a color palette formerly introduced. Cobalt(II) nitrate and methanolic solution of piperidine, or cyclohexylamine, respectively, were the suitable components to get unmistakable results.
Subject(s)
Urea/analogs & derivatives , Urea/analysis , Amines/chemistry , Color , Colorimetry , Dimethyl Sulfoxide , Indicators and Reagents , Reproducibility of Results , SpectrophotometryABSTRACT
An optical sensor system based on evanescent field excitation of fluorophore-labeled DNA-targets specifically binding to immobilized DNA probes has been developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disposable sensor chip via biotin/neutravidin linkage and hybridize to complementary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization rate was constant and directly proportional to the target concentration. When an 18mer oligonucleotide was used as a probe a linear calibration curve was obtained for a 56mer single-stranded DNA target derived from the neomycin phosphotransferase gene, a selection marker in a variety of genetically modified plants, with an estimated lower limit of detection of 0.21 nmol L(-1). No cross-hybridization to a 51mer actin DNA target was observed and even a single-nucleotide mismatch led to a negligible signal. A shutter in the readout device enabled separate detection of targets hybridizing to probes immobilized at the inlet and outlet sides, respectively, of the flow channel. This opens a route toward a real-time DNA array format with analysis times as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR product was measured after separation of the double-stranded DNA by simple heat denaturation with a detection limit clearly lower than that of traditional gel electrophoresis.
Subject(s)
Biosensing Techniques/instrumentation , DNA/analysis , Oligonucleotide Array Sequence Analysis/instrumentation , Actins/genetics , Biosensing Techniques/methods , Calibration , DNA Probes , DNA, Single-Stranded/analysis , Fluorescent Dyes , Humans , Kanamycin Kinase/genetics , Nucleic Acid Hybridization/methods , Oligonucleotide Array Sequence Analysis/methods , Sensitivity and SpecificityABSTRACT
PURPOSE: To evaluate the safety and effectiveness of gadodiamide-enhanced magnetic resonance (MR) angiography with single and triple doses in the assessment of abdominal arterial stenoses. MATERIALS AND METHODS: One hundred five patients were included in the randomized, double-blind, phase III multicenter trial. Results of MR angiography with 0.1 mmol/kg and 0.3 mmol/kg doses of gadodiamide were compared with those of digital subtraction angiography (DSA) and according to dose. RESULTS: No serious adverse events were observed. The mean contrast index at the region proximal to the primary stenosis was significantly higher in the triple-dose group (P =.03). Mean 95% CI values for the difference in depicted degree of stenosis between DSA and postcontrast MR angiography improved from -3.4% +/- 4.7 (SD) in the single-dose group to -1.2% +/- 4.7 in the triple-dose group. Mean values for overall image quality on the visual analogue scale improved with the triple dose (P =.02). Confidence in diagnosis was high at postcontrast MR angiography in 88% and 96% of cases in the single- and triple-dose groups, respectively. CONCLUSION: Gadodiamide-enhanced MR angiography performed with single and triple doses is safe and effective for assessing major abdominal arterial stenoses. Although high agreement between MR angiography and DSA was achieved with both doses, triple-dose MR angiography was superior in the evaluations of image quality, degree of arterial stenoses, and confidence in diagnosis.
Subject(s)
Abdomen/blood supply , Arterial Occlusive Diseases/diagnosis , Contrast Media/administration & dosage , Gadolinium DTPA , Image Enhancement , Magnetic Resonance Angiography , Magnetic Resonance Imaging , Aged , Aged, 80 and over , Angiography, Digital Subtraction , Contrast Media/adverse effects , Dose-Response Relationship, Drug , Double-Blind Method , Female , Gadolinium DTPA/administration & dosage , Gadolinium DTPA/adverse effects , Humans , Male , Middle Aged , Sensitivity and SpecificityABSTRACT
The aim of this study was to assess the accuracy of double-contrast magnetic resonance imaging (MRI) with rectal application of the superparamagnetic iron oxide contrast agent (SPIO) ferristene and IV gadodiamide for preoperative staging of rectal cancer. In a randomized phase II dose-ranging trial, 113 patients were studied preoperatively with one of four different formulations of ferristene (Abdoscan) as an enema before MRI. T1-weighted spin-echo (T1w SE) and T2w turbo spin-echo (TSE) single-contrast images were obtained as well as T1w SE and gradient-echo (GRE) double-contrast images after IV gadodiamide injection (Omniscan). Images were assessed qualitatively, and TNM tumor stage was compared with histopathology. High-viscosity ferristene formulations were superior to low-viscosity formulations in tumor staging (accuracy 90% vs 74%, P < 0.01). There was no significant difference between high and low iron content ferristene. MRI had a sensitivity of 97%, specificity of 50%, and accuracy of 82% for staging of rectal carcinoma higher than T2 stage. At receiver operator characteristic (ROC) analysis, MR differentiation between T1/T2 and T3/T4 tumor stages yielded a ROC index of 0.848. Double-contrast MRI is an accurate method for preoperative staging of rectal cancer.
Subject(s)
Adenocarcinoma/pathology , Contrast Media , Ferric Compounds , Gadolinium DTPA , Magnetic Resonance Imaging/methods , Rectal Neoplasms/pathology , Adenocarcinoma/diagnosis , Adenocarcinoma/surgery , Administration, Rectal , Adult , Aged , Biopsy, Needle , Chi-Square Distribution , Dose-Response Relationship, Drug , Female , Humans , Image Processing, Computer-Assisted , Injections, Intravenous , Male , Middle Aged , Neoplasm Staging/methods , Preoperative Care , Probability , ROC Curve , Rectal Neoplasms/diagnosis , Rectal Neoplasms/surgery , Sensitivity and SpecificityABSTRACT
BACKGROUND AND PURPOSE: Reconstruction of first-pass bolus information to derive regional cerebral blood volume (rCBV) maps is commonly performed in many centers; however, various protocols with different doses of paramagnetic contrast injections have been reported. We evaluated the dose dependency of rCBV maps in a brain tumor population by using three different doses of gadodiamide injection to evaluate their diagnostic accuracy in blinded reader sessions. METHODS: Eighty-three patients with intraaxial brain tumors (72 gliomas) were studied at three centers and randomized to receive a bolus injection of 0.1, 0.2, or 0.3 mmol/kg per body weight of gadodiamide. rCBV maps were generated from T2*-weighted gradient-echo echoplanar sequences at 1.5 T. Data processing was performed according to the indicator dilution theory. RESULTS: The mean contrast-to-noise ratio (CNR) was significantly different between gadodiamide doses of 0.1 and 0.2 mmol/kg (CNR = 8.7 and 15.7) and between 0.1 and 0.3 mmol/kg (CNR = 17.7). No significant difference was found between doses of 0.2 and 0.3 mmol/kg. Sensitivity for the differentiation of benign and malignant brain tumors was 80%, 95%, and 91%, and specificity was 45%, 54%, and 43% by blinded readings at 0.1, 0.2, and 0.3 mmol/ kg, respectively, as compared with histologic findings. Nonblinded readings had a sensitivity of 83%, 100%, and 90% and a specificity of 82%, 100%, and 73% at 0.1, 0.2, and 0.3 mmol/kg, respectively. CONCLUSION: A dose of 0.2 mmol/kg of gadodiamide is recommended for reconstruction of rCBV maps if data are acquired with the T2*-weighted protocol described.
Subject(s)
Blood Volume , Brain Neoplasms/physiopathology , Cerebrovascular Circulation , Contrast Media/administration & dosage , Gadolinium DTPA/administration & dosage , Magnetic Resonance Imaging , Brain Neoplasms/diagnosis , Double-Blind Method , Female , Humans , Injections, Intravenous , Male , Middle Aged , Sensitivity and SpecificityABSTRACT
Nowadays many reagents are available for the biotinylation of proteins. As most of them bind to amino groups of the protein the degree of labelling differs from batch to batch and the possibility exists that the biological activity of the target protein may be affected by the labelling procedure. In the present study we have investigated an enzymatic approach to biotinylation using microbial transglutaminase (MTGase) from Streptoverticillium mobaraense. The proposed method is particularly suitable when only a few biotin molecules need to be attached to the target proteins. The enzyme catalyses the acyl transfer reaction between gamma-carboxyamide groups and various primary amines. This was exploited for biotinylation using two amino-modified biotin derivatives, biotinamido-5-pentylamin (BIAPA) and biotinoyl-1,8-diamino-3, 6-dioxaoctane (BIDADOO) as acyl acceptors and a monoclonal IgG against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the acyl donor. Kinetic studies revealed that the MTGase-mediated reaction proceeds with low velocity and is almost complete after 34 h. Conjugation ratios ranging from 1.1 to 1.9 biotins per IgG were found by mass spectrometry. To investigate the influence of antibody conjugation on antigen binding a competitive ELISA for the determination of 2,4-D employing MTGase-biotinoylated IgGs was developed. In this assay lower limits of detection of 0.3 and 1.0 microg/l of 2,4-D were achieved with BIDADOO- and BIAPA-modified antibodies, respectively.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/immunology , Antibodies, Monoclonal/metabolism , Biotinylation/methods , Immunoglobulin G/metabolism , Transglutaminases/metabolism , 2,4-Dichlorophenoxyacetic Acid/analysis , Antibody Specificity , Biotin/analogs & derivatives , Enzyme-Linked Immunosorbent Assay , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Streptomycetaceae/enzymologyABSTRACT
An optical sensor system is described which is particularly well suited for medical point-of-care diagnostics. The system allows for all kinds of immunochemical assay formats and consists of a disposable sensor chip and an optical readout device. The chip is built up from a ground and cover plate with in- and outlet and, between, of an adhesive film with a capillary aperture of 50 microns. The ground plate serves as a solid phase for the immobilization of biocomponents. In the readout device, an evanescent field is generated at the surface of the ground plate by total internal reflection of a laser beam. This field is used for the excitation of fluorophor markers. The generated fluorescence light is detected by a simple optical setup using a photomultiplier tube. Because of the evanescent field excitation, washing or separation steps can be avoided. With this system the pregnancy hormone chorionic gonadotropin (hCG) could be determined in human serum with a detection limit of 1 ng/mL. Recovery values were 86, 106, and 102% for 5, 50, and 100 ng/mL hCG, respectively. The SD in repeated measurements (n = 10) was 5.6%. Furthermore, the feasibility of the system in competitive-type immunoassays was demonstrated for serum theophylline. A linear calibration curve of signal vs theophylline between 1 and 50 mg/L was obtained. Recovery values varied between 118% (10 mg/L) and 81.0% (20 mg/L).
Subject(s)
Biosensing Techniques/instrumentation , Diagnostic Techniques and Procedures/instrumentation , Chorionic Gonadotropin/blood , Female , Humans , Optics and Photonics , Pregnancy , Sensitivity and Specificity , Theophylline/analysisABSTRACT
The influence of antibody valency in a displacement immunoassay was investigated by comparing the whole antibody molecule with the corresponding Fab-fragment. The displacement immunoassay for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) takes advantage of the cross-reactivity of monoclonal anti-2,4-D antibodies and the Fab-fragments toward immobilized 2-methyl-4-chlorophenoxyacetic acid (MCPA). Due to the low affinity of the antibodies toward MCPA (cross-reactivity of approximately 30%), the addition of 2,4-D resulted in a displacement of the antibodies or the fragments. The detection limits obtained with whole anti-2,4-D antibodies and Fab-fragments were 0.1 microg/l and 0.01 microg/l 2,4-D, respectively. The whole antibodies and the Fab-fragments show similarities, such as the cross-reactivity toward MCPA (26% and 33%), and some characteristics of the calibration curve, for example the large detection range and the sensitivity. In contrast to the bivalent antibodies, however, increasing the hapten/protein ratios of the immobilized MCPA-BSA conjugates did not affect the detection limit when using the Fab-fragments. Moreover, kinetic experiments reveal a faster displacement reaction with the Fab-fragments. A disadvantage of using the Fab-fragments is the generation of lower absorbance values in the ELISA.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/metabolism , Antibodies/chemistry , Antibodies/metabolism , Immunoassay/methods , 2,4-Dichlorophenoxyacetic Acid/immunology , Antibodies/immunology , Antibody Affinity , Binding, Competitive/immunology , Calibration , Cross Reactions , Haptens/metabolism , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , KineticsABSTRACT
A fully automated stand-alone flow injection immunoanalysis (FIIA) device for the determination of cephalexin in milk is developed with a main focus on the investigation of the influence of the sample matrix. The system is based on principles of flow-through immunoassays and on sequential addition of the assay components to an immunoreactor. Protein G is immobilised on the surface of the immunoreactor serving as affinity matrix for the polyclonal anti-cephalexin antibodies. A cephalexin-alkaline phosphatase conjugate is mixed with the analyte-containing sample and binds in a competitve manner to the corresponding antibodies in the immunoreactor. After substrate addition enzymatically generated p-aminophenol is detected at a carbon electrode at +150 mV vs. Ag/AgCl. One assay cycle takes 16 min including regeneration of the immunoreactor. The large excess of protein G allows for more than 150 regenerations without significant loss of signal height. Due to the high specificity of the anti-cephalexin antibodies, other beta-lactam antibiotics like penicillin, amoxicillin and cloxacillin do not interfere in the measurements, even when added at 10 mg l-1. To deactivate alkaline phosphatase present in milk, samples are heat-treated for 3 min prior to measurements. Cephalexin recoveries from two milk samples are 90 and 110%. The detection limit in milk is 1 microgram l-1 (mean relative standard deviation of 3%), less than the maximum residue level of 4 micrograms per kg milk fixed for some beta-lactam antibiotics in the European Union. The device is suitable for fast quantitative data generation from consecutively measured samples and thus adds to analytical screening methods.
Subject(s)
Cephalexin/analysis , Cephalosporins/analysis , Drug Residues/analysis , Milk/chemistry , Animals , Flow Injection AnalysisABSTRACT
Taking the example of Nuremberg, a city of half a million inhabitants, the article illustrates how, despite very restricted resources, the local public health department was able to establish a series of health reports dealing with different topics. Treatment of a number of topics was only possible through cooperation with the local university. Initially two health reports were produced on a regional basis for the purpose of supporting health promotion in particular areas of the city. Whereas the first report attempts to give a comprehensive picture of health relevant living conditions in a certain area, the second report concentrates on an analysis of the situation of children and young people in a different part of the city. Another area of concern deals with the gathering of specific health related data. The data from a pediatric health sentinel in Nuremberg were combined with data on climate and air pollution in the years 1995 and 1996. Various dissertation topics were useful in the analysis of everyday public health data. Most of these results have been published in a separate reader. They include an analysis of the reports regarding pregnancy counciling, standardised reports of the HIV antibody tests as well as an analysis of the causes of infant mortality based on death certificates. Finally, a comparative study of basic health data from various cities is in progress. It is hoped that this will offer a basis for an evaluation of the health situation in Nuremberg.
Subject(s)
Population Surveillance , Public Health/statistics & numerical data , Urban Health/statistics & numerical data , Adolescent , Adult , Aged , Child , Child, Preschool , Data Collection , Female , Germany , Humans , Infant , Infant, Newborn , Male , Middle Aged , PregnancyABSTRACT
A new method based on enzymatic single-step in situ synthesis of hapten-carrier conjugates on electrodes is described yielding stable, reproducible, and reusable organic-phase immunoelectrodes (OPIEs). The electrodes developed were tailored for analyte detection in organic solvents and allow for the analysis of soil extracts without further sample processing and cleanup. Catalyzed by transglutaminase from a variant of Streptoverticillium mobaraense, the reaction proceeds in aqueous solution with and without addition of organic media in only 1.5 hours. In this study, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as model compound and chemically amino-functionalized prior to its enzymatic immobilization. The high reproducibility of the immobilization procedure allowed for batch calibration of the immunoelectrodes. Moreover, pure methanol or treatment with diluted sulfuric acid used for regeneration studies did not disturb the hapten layer. The OPIE consists of screen-printed carbon electrodes, monoclonal anti-2,4-D antibodies, and the immunochemical recognition reaction and was optimized with regard to a high stability in organic media. For electrochemical detection, horseradish peroxidase was used as enzyme label together with H2O2 as substrate and hexacyanoferrate (II)/(III) as mediator. The OPIE showed high stability upon storage over 93 days. Response times of 17 s (t95) were found to be advantageous compared to those of other biosensors. Including the immunochemical reactions, the complete assay takes 30 min. A calibration curve for 2,4-D in 30% methanol/buffer obtained with 70 electrodes within 4 weeks revealed a detection limit of 9 mg/L, a sensitivity of 1.3 nA L mg-1 cm-2, and a repeatability of 6.8%. Although we calculated a lowered repeatability for reused electrodes of 13.4% and a slightly decreased sensitivity of 0.9 nA L mg-1 cm-2, multiple-used OPIEs could also be applied for calibration.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/chemistry , Antibodies, Monoclonal/chemistry , Herbicides/chemistry , Immunoenzyme Techniques/instrumentation , Feasibility StudiesABSTRACT
RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the pharmacokinetics, dialysability, and safety of gadodiamide injection in patients with severely reduced renal function not treated with renal replacement therapy and patients undergoing hemodialysis or continuous ambulatory peritoneal dialysis. MATERIALS AND METHODS: Twenty-seven patients--nine with severely reduced renal function (glomerular filtration rate, 2-10 mL/min), nine undergoing hemodialysis, and nine undergoing continuous ambulatory peritoneal dialysis--were followed up for 5, 8, and 22 days, respectively, after receiving gadodiamide injection (0.1 mmol per kilogram body weight). RESULTS: Gadodiamide injection caused no changes in renal function. In patients with severely reduced renal function, the elimination half-life of gadodiamide injection was prolonged (34.3 hours +/- 22.9) compared with data in healthy volunteers (1.3 hours +/- 0.25). An average of 65% of the gadodiamide injected was eliminated during a hemodialysis session. After 22 days of continuous ambulatory peritoneal dialysis, 69% of the total amount of gadodiamide was excreted; this reflects the low peritoneal clearance. In all patients, no metabolism or transmetallation of gadodiamide was found. There were no contrast material-related adverse events. CONCLUSION: Gadodiamide is dialysable and can safely be used in patients with severely impaired renal function or those undergoing hemodialysis or continuous ambulatory peritoneal dialysis. No precautions to increase the elimination are necessary.
Subject(s)
Contrast Media/pharmacokinetics , Gadolinium DTPA/pharmacokinetics , Peritoneal Dialysis, Continuous Ambulatory , Renal Dialysis , Renal Insufficiency/metabolism , Adult , Aged , Chromatography, High Pressure Liquid , Female , Follow-Up Studies , Half-Life , Humans , Injections, Intravenous , Male , Middle Aged , Renal Insufficiency/therapyABSTRACT
Hapten-protein conjugates are essential in many immunochemical assays, in particular, in assays employing titration or competitive assay formats. By exploitation of the catalytic properties of the microbial transglutaminase from Streptoverticillium mobarense sp. (MTGase), i.e., acyl transfer between gamma-carboxamide groups and various primary amines, new techniques for the synthesis of hapten-protein conjugates were developed. This is demonstrated by two examples. The feasibility of MTGase for hapten-protein conjugate synthesis was studied by coupling the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) to casein. Different procedures for the synthesis and the immobilization of these 2,4-D-casein conjugates were evaluated, comprising (i) a batch procedure, (ii) coupling of 2,4-D to an already immobilized layer of casein, and (iii) a method for simultaneous immobilization and conjugation. Kinetic studies revealed that conjugate formation in the batch procedure was almost complete after approx 2 h. By employing the conjugates in a competitive ELISA, detection limits as low as 0.05 microgram/L 2,4-D were reached. Using the approach with simultaneous immobilization and conjugation, the time for the whole assay could be reduced to only 2 h. Finally, to demonstrate the versatility of the enzymatic synthesis of hapten-protein conjugates, an ELISA for 2,4,6-trinitrotoluene (TNT) determination based on transglutaminase-synthesized conjugates was developed. In this assay, a detection limit as low as 0.04 microgram/l TNT was obtained.
Subject(s)
Caseins/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Haptens/chemistry , Transglutaminases/metabolism , 2,4,5-Trichlorophenoxyacetic Acid/analysis , 2,4-Dichlorophenoxyacetic Acid/analysis , Catalysis , Streptomycetaceae/enzymologyABSTRACT
An enzyme-linked immunosorbent assay-based displacement assay was developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Advantage was taken of the cross-reactivity of a monoclonal anti-2,4-D antibody toward 2-methyl-4-chlorophenoxyacetic acid (MCPA). MCPA was conjugated with bovine serum albumin (BSA), immobilized on the surface of a microtiter plate, and saturated with the anti-2,4-D antibody. Due to the low affinity of the antibody toward MCPA (cross-reactivity of approximately 30%), the addition of 2,4-D resulted in a displacement of the antibody. Remaining antibodies were subsequently detected using a peroxidase-labeled goat anti-mouse antibody. The detection limit was as low as 0.1 microgram/liter for 2,4-D, which complies with the European Union Drinking Water Directives. When 2,4-D-BSA was used instead of MCPA-BSA conjugates, no significant displacement of bound antibody was observed.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/analysis , Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/methods , 2,4-Dichlorophenoxyacetic Acid/immunology , 2-Methyl-4-chlorophenoxyacetic Acid/analysis , 2-Methyl-4-chlorophenoxyacetic Acid/chemistry , 2-Methyl-4-chlorophenoxyacetic Acid/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Cross Reactions , Haptens/analysis , Haptens/immunology , Indoleacetic Acids/chemistry , Serum Albumin, Bovine/chemistryABSTRACT
Ultrafast contrast-enhanced magnetic resonance imaging can be used to distinguish open and closed infarct-related arteries. An open artery is characterized by a faster rise and fall in signal intensity.