A binding protein displaying broad-spectrum cross-reactivity within the sulfonamide group was used in conjunction with a sulfonamide specific sensor chip and a surface plasmon resonance biosensor to develop a rapid broad spectrum screening assay for sulfonamides in porcine muscle. Results for 40 samples were available in just over 5h after the completion of a simple sample preparation protocol. Twenty sulfonamide compounds were detected. Acetylated metabolites were not recognised by the binding protein. Limit of detection (mean–three times standard deviation value when n=20) was calculated to be 16.9ngg−1 in tissue samples. Intra-assay precision (n=10) was calculated at 4.3 %CV for a sample spiked at 50ngg−1 with sulfamethazine, 3.6 %CV for a sample spiked at 100ngg−1 with sulfamethazine, 7.2 %CV for a sample spiked at 50ngg−1 with sulfadiazine and 3.1 %CV for a sample spiked at 100ngg−1 with sulfadiazine. Inter-assay precision (n=3) was calculated at 9.7 %CV for a sample spiked at 50ngg−1 with sulfamethazine, 3.8 %CV for a sample spiked at 100ngg−1 with sulfamethazine, 3.5 %CV for a sample spiked at 50ngg−1 with sulfadiazine and 2.8 %CV for a sample spiked at 100ngg−1 with sulfadiazine.

Two surface plasmon resonance (SPR)-based biosensor assays for detection of beta-lactam antibiotics in milk are reported. The assays are based on the enzymatic activity of a carboxypeptidase converting a 3-peptide into a 2-peptide, a reaction that is inhibited in the presence of beta-lactams. Antibodies were used to measure either the amount of formed enzymatic product or the amount of remaining enzymatic substrate. Both assays detected different beta-lactams at or below European maximum residue limits (MRLs), and the detection limit for penicillin G was 1.2 microg/kg and 1.5 microg/kg for the 2- and 3-peptide assays, respectively. The precision (CV) was < 5%, both within and between assays at the penicillin G MRL (4 microg/kg). The biosensor results obtained upon analysis of incurred milk samples were compared with results obtained by liquid chromatography (HPLC), and the method agreements were, in general, good.

The beta-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest.A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect beta-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact beta-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of beta-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 mug kg(-1) for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 mug kg(-1). The coefficient of variation at 4 mug kg(-1) penicillin G, ranged between 7.3 and 16% on three different days. (C) 2002 Elsevier Science B.V. All rights reserved.

An optical biosensor assay for detection of beta-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a beta-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by beta-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the beta-lactam concentration of the sample. The detection limit for a number of commonly used beta-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 mg kg(-1). For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.

Clenbuterol (CBL) is an orally active beta2-adrenoceptor agonist which has been used in veterinary medicine as a broncodilator and an agent of uterine relaxation. It has however become better known as a drug used illegally to promote growth in farm animals. A rapid and sensitive biosensor assay was developed to detect CBL residues in bovine urine. The method involved a simple extraction procedure using tert-butyl methyl ether followed by analysis on the biosensor with results obtained against a buffer calibration curve. The assay allowed up to 88 samples to be analyzed per working day, with each cycle on the biosensor taking approximately 7 min to complete. The limit of detection (LOD) was determined as 0.27 ng/mL using 20 EU reference blank urine samples. The intra-assay Sr ranged from 4.7-7.6% for 3 control samples while the interassay Sr ranged from 9.2-12.7%. The recovery was found to be approximately 95%. A series of incurred urine samples were assayed and the results compared by Enzyme immunoassay (EIA), radio-immunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS) analysis. Urine samples taken from local abattoirs were also analyzed by the biosensor method and by EIA analysis. The antibody used in the biosensor test exhibited high cross reactivity with at least 7 other beta-agonists allowing detection of these compounds at less than 1 ng/mL in bovine urine.

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.

In three patients with carcinoid liver and/or lymph node metastases, we studied the process of tumor tracer uptake and decarboxylation by means of positron emission tomography (PET) using 5-hydroxy-L-tryptophan (5-HTP) 11C-labeled in the β-position (HTP) and later the same day with 5-HTP 11C-labeled in the carboxyl group (HTC). With HTP, in which the 11C-label follows the molecule through decarboxylation to form 11C-serotonin, a high tumor accumulation of the tracer was found. With HTC, in which the label is rapidly eliminated from the tissues as 11CO2 if decarboxylation takes place, there was virtually no uptake by the tumors. By utilizing data from PET scanning with both tracers, we could quantify the decarboxylation rate and tissue accumulation of [11C]-serotonin and hence the enzymatic action of aromatic amino acid decarboxylase.

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 mu g kg(-1) and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cow's milk. The advantages of the new assay format include analyse-independent immobilisation and regeneration. Furthermore, the assay enables measurements with covalent interactions between analyte and detecting molecule. The main disadvantage is the requirement of a conjugate between analyte and the hapten H1. Moreover, it is likely that the antibody surface will have a shorter life span than a surface with the antimicrobial immobilised.

PURPOSE Carcinoid tumors, especially those of midgut origin, produce serotonin via the precursors tryptophan and 5-hydroxytryptophan (5-HTP). We have evaluated the usefulness of positron emission tomography (PET) with carbon-11-labeled 5-HTP in the diagnosis and treatment follow-up evaluation of patients with neuroendocrine tumors. PATIENTS AND METHODS PET using 11C-labeled 5-HTP was compared with computed tomography (CT) in 18 patients (14 midgut, one foregut, one hindgut carcinoid, and two endocrine pancreatic tumors [EPT]). In addition, 10 of 18 patients were monitored with PET examinations during treatment. RESULTS All 18 patients, including two with normal urinary 5-hydroxyindole acetic acid (U-5-HIAA), had increased uptake of 11C-labeled 5-HTP in tumorous tissue as compared with normal tissue. Liver metastases, as well as lymph node, pleural, and skeletal metastases, showed enhanced 5-HTP uptake and PET could detect more lesions than CT in 10 patients and equal numbers in the others. Tumor visibility was better for PET than for CT due to the high and selective uptake of 5-HTP with a high tumor-to-background ratio. Binding studies indicated an irreversible trapping of 5-HTP in the tumors. Linear regression analyses showed a clear correlation (r = .907) between changes in U-5-HIAA and changes in the transport rate constant for 5-HTP during treatment. CONCLUSION PET with 11C-labeled 5-HTP demonstrated high uptake in neuroendocrine gastrointestinal tumors and thereby allowed improved visualization compared with CT. The in vivo data on regional tumor metabolism, as expressed in 11C-5-HTP uptake and transport rate, provided additional information over conventional radiologic techniques. The close correlation between the changes in 11C-5-HTP transport rate and U-HIAA during medical treatment indicates the potential of 11C-5-HTP-PET as a means to monitor therapy.

The present study was undertaken to investigate if pretreatment with pharmacological agents could change the organ uptake of 11C-labelled l-DOPA, and especially if the urinary excretion could be decreased. l-[β-11C]DOPA was injected IV into unanesthetized Sprague-Dawley rats. After 20 min the rats were decapitated and organs taken out for radioactivity measurements. The uptake in the organs was investigated in animals only given the tracer, and in animals pretreated with drugs such as decarboxylase inhibitors carbidopa and benserazide as well as the monoamine oxidase inhibitors deprenyl, clorgyline, and the COMT inhibitor OR-486. A marked decrease in the urinary radioactivity was observed after carbidopa and benserazide administration. HPLC analysis revealed that under native conditions the major part of urinary radioactivity existed as dopamine, which was eliminated by the decarboxylase inhibitors. After pretreatment with the COMT inhibitor OR-486, the radioactivity uptake in the pancreas increased fourfold as compared to non-treated animals. HPLC analysis showed that this correlated with a marked increase in radiolabelled DOPAC. In the other organs and with the other drugs, only small effects were observed. With l-[β-11C]fluoroDOPA as a tracer, similar results were observed although the increase in the pancreas by OR-486 had a lower magnitude. These studies suggest that it might be possible to improve the diagnostic ratio of l-[β-11C]DOPA or l-[18F]fluoroDOPA in whole-body PET studies by pretreating the patient with decarboxylase inhibitor for reducing the urinary excretion and potentially increase the target organ uptake by COMT inhibition.

The regional brain distribution and selective binding of the cholinergic muscarinic receptor agonist CI-979 labelled with 11C was studied in rhesus monkeys by means of positron emission tomography. The selective binding was measured as displacement of [11C]CI-979-derived radioactivity following constant-rate infusion of CI-979 at doses of 0.5-10 micrograms/kg/h. An extensive and rapid distribution of [11C]CI-979 was observed to the basal ganglia and temporal occipital cortices, i.e., regions of the brain with a high density of muscarinic receptors. The radioactivity was dose-dependently decreased in cortical regions following infusions of unlabelled CI-979 (0.5-10 micrograms/kg/h), indicating selective receptor binding of [11C]CI-979 in these brain regions. The binding of [11C]CI-979 was unaltered or even higher in the striatum following unlabelled CI-979 infusion. The high densities of autoreceptors in the striatum may explain the inhibitory feedback mechanism on endogenous acetylcholine induced by the muscarinic receptor agonist. Since muscarinic receptor agonists release acetylcholine, the higher releasable pool of acetylcholine in the stratum will induce feedback inhibition of acetylcholine release and less competition between acetylcholine and CI-979 at the muscarinic receptors. This may explain the differences between cortex and striatum in [11C]CI-979 binding. The cerebral blood flow was not changed by the infusion of unlabelled CI-979, supporting the assumption that effects of CI-979 in brain may be due to the interaction with brain muscarinic receptors. Despite a relative rapid clearance of [11C]CI-979 radioactivity from binding in the brain characteristic of a receptor agonist, the radioligand might be useful in positron emission tomography studies to reveal changes in cholinergic receptors and functional activity in Alzheimer patients treated with muscarinic agonists.

A high performance liquid chromatographic system was developed for separation of 11C-labeled 5-hydroxy-L-tryptophan ([11C]HTP) and in vivo formed radiolabeled metabolites in rat brain tissue. Analysis of brain homogenate revealed that the main part of the radioactivity was associated with 11C-labeled 5-hydroxyindole-3-acetic acid after intravenous injection of [11C]HTP to the rat. The serotonin synthesis rate in the brain was calculated and closely correlated to the serotonin synthesis rate in monkey and human measured using positron emission tomography.

Positron emission tomography (PET) was used in a multitracer protocol to evaluate D-amphetamine induced effects on dopamine biosynthesis rate and release in propofol anesthetized Rhesus monkeys.l-[β-11C]DOPA was used as biochemical probe to study the brain dopamine biosynthesis rate whilst dopamine release was followed by the binding displacement of the [11C]-radiolabelled dopamine receptor antagonists, raclopride and N-methylspiperone. Studies were performed with either a constant rate intravenous infusion of D-amphetamine aiming at plasma concentrations of 0.2 to 25 ng/ml or with intravenous bolus doses of 0.1 and 0.4 mg/ kg. Decreased binding of the dopamine receptor antagonists was measured in both modes of D-amphetamine administration but notably [11C]N-methylspiperone was less able to sense D-amphetamine induced release of dopamine. At plasma concentrations aimed above 1 ng/ml a levelling off of the binding of [11C]raclopride at 68 ± 8.1% of the baseline value indicated that displacement was only possible from a fraction of the binding sites. Amphetamine was observed to increase the rate constant forl-[β-11C]DOPA utilization in the brain. This was most likely due to an acutely induced subsensitivity of presynaptic dopamine receptors.l-[β-11C]DOPA and [11C]raclopride were found suitable to indicate changes in dopamine synthesis rate and release respectively using PET and can be used to mirror drug-induced changes of brain dopaminergic function.

Significant increases in striatal L-[C-11]DOPA retention were observed in adult female rhesus monkeys with positron emission tomography (PET) following administration of drugs that increase cerebral L-DOPA concentrations. The monkeys were scanned twice: at baseline (using 10-50 mu g of tracer substance) and during continuous administration of L-DOPA (3 or 15 mg/kg/h) and 6-R-Erythro-4,5,6,7-tetrahydrobiopterin (6R-BH4) (5 mg/kg/h) and during combined administration of both drugs. PET scans of L-[C-11]DOPA distribution were obtained in GE2048-15B or GE4096-15WB Plus positron tomographs. In all studies the specific striatal L-[C-11]DOPA influx rate increased by an average of 17-20%. These increases were significantly higher than the retest variability obtained with saline infusions under identical experimental conditions. In individual monkeys the magnitude of increase in the striatal L-[C-11]DOPA influx rate varied from no effect of the drug infusion to a 45% increase. Taken together, the results of this study demonstrate that L-DOPA in itself can affect dopaminergic neurotransmission in vivo and also adds further evidence that the neuromodulatory effects of the amino acid are predominantly autoreceptor antagonist-like, The findings most likely have importance for the further understanding of the dopaminergic system in neurodegenerative and psychiatric disorders. (C) 1997 Wiley-Liss, Inc.

As a complement to in vivo studies with positron emission tomography (PET), it is desirable to perform in vitro characterization of newly developed 11C tracers. In this report we describe the technique for determination of receptor-ligand kinetics utilizing ligands labeled with the short-lived radionuclide 11C. The limitations and advantages are discussed. The benzodiazepine antagonist [11C]Ro 15-1788 was used as a model substance, and the use of storage phosphor plates for quantification of radioactivity was validated. Storage phosphor plates showed an excellent linear range (~103) and acceptable resolution (~ 0.5 mm). Receptor-ligand kinetics, including depletion, association and dissociation, saturation and displacement were evaluated with good results through the use of short-lived radiotracers and storage phosphor plates.

Positron emission tomography (PET) or, with another interpretation, positron emitting tracers, is a tracer technique which provides a new means for measuring biological functions or biochemistry in vivo in research animals and humans. The technique utilizes short lived accelerator produced radionuclides, typically C-11, N-13, O-15 and F-18, with half-lives of 20.3, 10, 2 and 110 min, respectively. These radionuclides can be incorporated position specifically into a variety of tracer molecules. Upon PC emission, two annihilation photons are emitted at 180 degrees from each other, making it possible to measure quantitatively the radioactivity concentration, for example by a positron camera. Since some of these are radionuclides of elements common in biomolecules, the potential is very great for developing many tracer molecules by synthetic labelling chemistry. They are all produced by nuclear reactions using charged particle accelerators with protons or deuterons. Despite the short half-life, synthetic methods and techniques are available to produce radiopharmaceuticals labelled with some of these radionuclides in a controlled way, allowing routine production of a large number of radiotracers with application potential in many areas. For example, starting with the nuclear reaction N-14(p, a)C-11 using a proton beam on a nitrogen gas target, labelled C-11-carbon dioxide can be produced in high specific radioactivity of the order of several Ci/mu mol, which allows radiotracer applications in vivo at very low concentrations. The selection of the tracers has of course to be made in relation to the question addressed. The tracers can on some occasions be specifically designed to interpret the obtained PET data in a quantitative manner or to validate the models used in the quantification. Here, considerations regarding stereochemistry, the position of the label or the use of multiple labelling can give the most valuable information. The high specific radioactivity and the short half-lives allow the design of experimental protocols utilizing combinations of such short lived positron emitting tracer molecules in multitracer studies. In the paper some of the above mentioned points are discussed and examples given to illustrate that PET is really a technique of using positron emitting tracer molecules.

In vivo demonstration of enzyme activity in endocrine pancreatic tumors - decarboxylation of 11C-dopamine.

Positron emission tomography (PET) with C-11- 5-hydroxytryptophan is used for the diagnosis in patients with neuroendocrine tumors. In these examinations, a disturbingly high uptake of the tracer is seen in the kidney pelvis and urinary bladder. The present study was undertaken to investigate if administration of pharmacological agents could change the relative uptake between organs. The tracer 5-hydroxy-L-[beta-C-11]tryptophan (HTP) was injected into rats. After 20 minutes they were decapitated and organs taken out for radioactivity measurements. One kidney was processed for ex vivo autoradiography. The radioactivity uptake was investigated in animals only given the tracer and in animals given the decarboxylase inhibitors carbidopa and benserazide, the monoamine oxidase inhibitors clorgyline and deprenyl or the inhibitors of tubular transport probenecide and cimetidine.With decarboxylase inhibition, a significant increase of tracer accumulation was observed especially in the pancreas whereas the radioactivity in urine decreased by a factor of 5. HPLC separation of urine revealed that 64 % of the radioactivity was in the form of C-11-serotonin. Clorgyline more than doubled the uptake in the pancreas but had no effect on the urinary excretion.The study demonstrates that significant reductions of urinary excretion can be obtained by decarboxylase inhibitors. Furthermore, the uptake in a target organ such as the pancreas can be significantly increased. These possibilities for inducing a redistribution of the tracer between organs might be used for enhancing the iii diagnostic potential of C-11-HTP.

The effect of 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) and l-tyrosine infusion on [11C]dopamine synthesis was analyzed in the striatum of Rhesus monkey using positron emission tomography (PET). The rate for decarboxylation from l-[β-11C]DOPA to [11C]dopamine was calculated using a graphical method with cerebellum as a reference region. Although the peripheral administration of 6R-BH4 at low dose (2 mg/kg) did not provide a significant increase in the rate of dopamine biosynthesis, a high dose of 6R-BH4 (20 mg/kg) induced an elevation of the rate. This 6R-BH4-induced elevation of the dopamine synthesis rate was further dose-dependently enhanced by the continuous infusion of l-tyrosine (0.2 and 1.0 μmol/min/kg). l-Tyrosine infusion with a rate of 1.0 μmol/min/kg caused an enhancement of the rate even during low dose administration of 6R-BH4 (2 mg/kg). l-Tyrosine infusion alone did not induce any elevation of the dopamine biosynthesis rate. The analysis of plasma indicated that the metabolic ratios of l-[β-11C]DOPA to each metabolite were not affected by 6R-BH4 and/or l-tyrosine infusion. The results suggest that the low dose loading of tyrosine facilitates the activity of 6R-BH4 on the presynaptic dopamine biosynthesis, and also that the combined effects can be monitored by PET using l-[β-11C]DOPA as a biochemical probe.