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At least 1,099 records · Page 61Linked to original sources
PubMed1985

A novel approach for the study of thromboxane A2 and prostaglandin H2 receptors using an 125I-labeled ligand.

Previous studies of eicosanoid receptors have utilized 3H-labeled ligands. However, 125I has a higher theoretical specific activity (approximately 2000 Ci/mmole) than 3H (29 Ci/mmole), which provides a potential advantage for 125I ligand binding studies when the receptor density is low. Since eicosanoids do not possess an easily iodinatable structure (e.g. a phenol or imidazole ring), it is not feasible to directly incorporate 125I into the molecule. The thromboxane A2/prostaglandin H2 receptor antagonist, cis-7-(2-p-hydroxyphenylethanolaminocyclopentyl)-heptanoic acid (cis-APO), was synthesized to test the concept that it could be labeled with 125I and used as a ligand for binding studies. cis-APO is a structural analog of 13-azaprostanoic acid, a TXA2/PGH2 antagonist [G. C. Le Breton, D. L. Venton, S. E. Enke and P. V. Halushka, Proc. natn. Acad. Sci. U.S.A. 76, 4097 (1979)], in which the omega aliphatic chain was substituted with 2-p-hydroxyphenylethanol, which contains a phenolic group. [127I]cis-APO was synthesized by insertion of 127I (stable isotope) into the phenolic portion of the molecule under alkaline conditions. [125I]-cis-APO was synthesized via insertion of 125I (unstable isotope) into the molecule in the presence of chloramine T. cis-APO inhibited human platelet aggregation induced by the thromboxane mimetic U46619 [C. Malmsten, Life Sci. 18, 169 (1976)]. The IC50 for cis-APO was 6.4 +/- 0.7 microM and for [127I]-cis-APO was 9.8 +/- 1.3 microM (P less than 0.001). [125I]-cis-APO binding to a human platelet membrane preparation at 4 degrees was time and protein concentration dependent, saturable, and reduced or abolished by trypsin or boiling respectively. The Kd for iodo-cis-APO determined at equilibrium using a Scatchard analysis was 1.48 microM and the maximum binding capacity was 18.7 pmoles/mg protein. The forward rate constant (k+1) was 2.3 X 10(3) M-1 s-1 and the dissociation constant (k-1) was 2.12 X 10(-3) s-1. The Kd determined from k-1/k+1 was 0.92 microM. These observations show that the omega side chain of eicosanoid analogs can be substituted with a phenolic group, iodinated, and retain biological activity. These molecules may then be utilized to study thromboxane A2 or prostaglandin H2 receptors.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1983

Radioimmunoassay of a glycoprotein associated with malignancy.

A glycoprotein associated with malignancy was purified from the 0.6M perchloric acid-soluble fraction of serum obtained from cancer patients. The purified glycoprotein contained sialic acid, which was responsible for binding to wheat-germ agglutinin-Sepharose. Gel electrophoresis showed one band with an apparent Mr of 50 000-55 000, and the isoelectric point was 4.4 +/- 0.1. The glycoprotein could be distinguished from carcinoembryonic antigen and alpha-fetoprotein. Iodination of this material with chloramine-T permitted development of a radioimmunoassay.

Glycoproteins↗
PubMed1987

Toxicity, mutagenicity, intracellular drug concentration and DNA binding in Escherichia coli treated with cis-platinum(II) complexes.

The genotoxic effects of six cis-platinum(II)chloramine complexes with different alkyl substituents on their amine ligands have been measured using Escherichia coli. The toxicity and mutagenicity of these compounds were compared, after exposure of bacteria, to drug concentrations which gave known quantities of platinum-DNA lesions. The results permit several observations concerning structure-activity relations of platinum(II) complexes. Firstly, methyl substitution on the amine ligands of cis-diamminedichloro-platinum(II) (DDP) is reported to reduce its antitumor activity. The methyl group did not exert an effect in bacteria where the toxicity and mutagenicity of cis-bis(methylamine)dichloroplatinum(II) and DDP were equivalent. In fact, at equal levels of DNA binding, complexes with substituted amines were generally more toxic toward bacteria than DDP. Secondly, replacement of the chloro groups of DDP by nitrato ligands increased its toxicity and mutagenicity at a given level of DNA binding. Hence, although DDP and its dinitrato derivative have identical ammine ligands, they may form different platinum-DNA lesions in bacteria. Finally, cis-bis(cyclohexylamine)-dichloroplatinum(II) was unique among the compounds studied since it did not cause bacterial filamentation or mutagenesis. These results suggest that, although this compound binds to the bacterial genome, it may not induce the SOS response.

DNA, Bacterial↗
PubMed1991

Evaluation of the ability of the angiotensin-converting enzyme inhibitor captopril to scavenge reactive oxygen species.

Captopril, an inhibitor of angiotensin-converting enzyme, has been suggested to have additional cardioprotective action because of its ability to act as an antioxidant. The rates of reaction of captopril with several biologically-relevant reactive oxygen species were determined. Captopril reacts slowly, if at all, with superoxide (rate constant less than 10(3) M-1 s-1) or hydrogen peroxide (rate constant less than M-1 s-1). It does not inhibit peroxidation of lipids stimulated by iron ions and ascorbate or by the myoglobin/H2O2 system. Indeed, mixtures of ferric ion and captopril can stimulate lipid peroxidation. Captopril reacts rapidly with hydroxyl radical (rate constant greater than 10(9) M-1 s-1) but might be unlikely to compete with most biological molecules for OH because of the low concentration of captopril that can be achieved in vivo during therapeutic use. Captopril did not significantly inhibit iron ion-dependent generation of hydroxyl radicals from hydrogen peroxide. By contrast, captopril is a powerful scavenger of hypochlorous acid: it was able to protect alpha 1-antiproteinase (alpha 1 AP) against inactivation by this species and to prevent formation of chloramines from taurine. We suggest that the antioxidant action of captopril in vivo is likely to be limited, and may be restricted to protection against damage by hypochlorous acid derived from the action of neutrophil myeloperoxidase.

Animals↗
PubMed1993

Improved synthesis of (5Z)-7-(3-endo-[(benzenesulfonamido)-bicyclo[2.2.1]heptyl]hept-5-enoic acid (S-145) derivatives and their iodine-125-labeled radioligands for the study of thromboxane A2 receptor.

An improved synthetic scheme for (5Z)-7-(3-endo-[(benzenesulfonamido)-bicyclo[2.2.1]heptyl)-h ept-5-enoic acid (S145) and its analogs has been designed. The procedure involves direct sulfonylation of 2-allyl-3-aminobicyclo[2.2.1]heptane intermediate followed by ozonolysis and addition of a C5 carboxyl unit. The yield of the final product was significantly improved. (5Z)-7-(3-endo-[(4-iodobenzenesulfonamido)-bicyclo [2.2.1]heptyl)hept-5-enoic acid (HS-145) and (5Z)-7-(3-endo-[(4-hydroxy-benzensulfonamido)-bicyclo [2.2.1]heptyl)hept-5-enoic acid (HS-145) were synthesized directly without any protection and deprotection steps. [125I](5Z)-7-(3-endo-[(4- iodobenzensulfonamido)-bicyclo[2.2.1]heptyl)hept-5-enoic acid ([125I]HS-145) was prepared from IS-145 through an organotin intermediate and [125I]sodium iodide with high specific radioactivity and good recovery of radioactivity. [125I](5Z)-7-(3-endo-[(4-hydroxy 3-iodo-benzenesulfonamido)-bicyclo[2.2.1]-heptyl)hept-5-enoic acid ([125I]HS-145) was prepared by direct iodination with sodium iodide using a modified chloramine-T method. Both [125I]HS-145 and [125I]HS-145 were found to be valuable radioligands for studying thromboxane A2 (TXA2) receptor.

Blood Platelets↗
PubMed1975

Production and storage of (125-I) thyroxine and (125-I) triiodothyronine of high specific activity.

High specific activity [125-I] triiodothyronine and [125-I] thyroxine have been produced regularly by the chloramine T radioiodination method. Simultaneous production of [125-I] triiodothyronine and [125-I] thyroxine is usual when diiodothyronine or triiodothyronine are employed as the starting materials. Specific activities reached vary with the starting compound (diiodothyronine, triiodothyronine, thyroxine) used, as both substitution and, less readily, exchange of iodine atoms take place. Starting with diiodothyronine specific activities of approximately 2400 and 5200 Ci/g were achieved for [125-I]triidothyronine and [125I] thyroxine, respectively, and, similarly, specific activities of approximately 1200 and 4000 Ci/g for [125I] triiodothyronine and [125I]-thyroxine, respectively, were reached when triiodothyronine was the starting material. [125-I] Triiodoacetic acid and [125I] tetraiodoacetic acid have been produced in the same way from triiodoacetic acid. By column chromatography on Sephadex G-25 (fine), eluting with alkaline phosphate buffer, good separation of the radioiodinated products has been readily achieved. Studies on the stability of the radioiodinated hormones showed that 50% methanol, ethanol, propanol and propylene glycol were all equivalent as preserving agents and, further, that the stability of the radioiodinated hormones was linearly related to the concentration of these preserving agents.

Chromatography, Gel↗
PubMed1980

Improved radioimmunoassay for human TSH.

This study concerns the optimization of the human TSH (h-TSH) radioimmunoassay with special emphasis on reducing the heterogeneity of the 125I h-TSH tracer. Enzymatic iodination of h-TSH with glucose oxidase/lactoperoxidase was shown to be superior to either low or high dose chloramine-T procedures, producing a high specific activity reagent (70--150 microCi/microgram) with minimal evidence of damage. Tracer purification procedures not only affected initial immunoactivity but also storage stability and heterogeneity of the resulting 125I h-TSH. Tracers purified by combining concanavalin A-Sepharose adsorption and high resolution gel filtration (Sephadex G100), produced significantly lower (p less than 0.001) serum h-TSH measurements than were observed in less purified tracer materials. Concanavalin A-Sepharose adsorption yield of the 125I h-TSH iodination products closely correlated with the yield (r = 0.85, p less than 0.001) and immunoactivity (r = 0.90, p less than 0.001) of the tracer produced, thus making this an ideal method for initial tracer purification. Storage of tracer adsorbed to a solid support (concanavalin A-Sepharose) reduced technical manipulations without compromising tracer performance. Loss of specific activity was minimized by storage at -70 degrees C. The assay developed using these technical approaches showed a sensitivity limit of 0.005 +/- 0.001 (S.E.M.) microU/tube; 50% displacement at 0.18 +/- 0.08 (S.E.M.) microU/tube and complete delineation between euthyroid (n = 49, 2.44 +/- 0.18 (S.E.M.) mU/l, range 1.00--6.08) and hyperthyroid (n = 62, 0.34 +/- 0.02 (S.E.M.) mU/l, range 0.10--0.85), serum h-TSH levels.

Chromatography, Gel↗
PubMed1981

Erythrocyte spectrin in Duchenne muscular dystrophy.

The primary structure of erythrocyte spectrin bands I and II from controls and patients with Duchenne muscular dystrophy was compared by 2-dimensional peptide mapping. 125I-labelling was done either by the chloramine-T method or using the Bolton and Hunter reagent followed by treatment with trypsin or chymotrypsin, resulting in four different peptide maps from each band of spectrin. Although all the peptide maps of band I were considerably different to those of band II, there were no consistent differences in the maps of bands I and II from controls compared to the corresponding maps from patients with Duchenne muscular dystrophy.

Chymotrypsin↗
PubMed1982

The purification and characterization of radiolabelled parathyroid hormone for radioimmunoassay.

An improved method for the purification of 125I-labelled PTH is presented. Following iodination of the hormone by the chloramine-T method, the reaction mixture is transferred directly to a 0.9 x 9 cm column of carboxymethyl-cellulose. Two major peaks emerge with the ammonium acetate gradient. Peak I represents labelled intact bPTH, Mr 9600, of good quality, i.e. a high B0 and a low NSB in the radioimmunoassay. Peak II, Mr approximately 12000-18000, gives a high B0, but also a high NSB and is less suitable for RIA. Peak II is not the result of iodination damage, as it is also present in the unlabelled PTH (Inolex); it is suggested that it represents either a pre-prohormone or an aggregate of PTH. The label produced is usable for up to 3 months. Trasylol and benzamidine protect the label against proteolytic degradation equally well; the latter, however, is considerably cheaper.

Benzamidines↗
PubMed1982

Double-antibody solid-phase radioimmunoassay for blood bradykinin.

A solid phase radioimmunoassay for the determination of blood bradykinin has been developed. Highly specific antibodies against bradykinin were raised in rabbits after coupling the peptide to thyroglobulin. Iodination of [Tyr8]-bradykinin was carried out with a chloramine-T procedure resulting in a tracer with high specific activity. Bradykinin was isolated in the following way: blood was sampled directly into acetone, and lipids were removed by extraction with petroleum either (40-60 degrees C). The final purification was made on QAE-Sephadex A-25 at pH 7.4. The mean recovery of added [125I-Tyr8]-bradykinin was 28% with a sample volume of 6 ml whole blood. The sensitivity of the radioimmunoassay was 1.25 pg/tube or 3 pg/ml blood. The reproducibility of the method is satisfactory with a between-assay coefficient of variation below 16%. Levels found in venous blood were below 3 pg bradykinin/ml in normal persons.

Adult↗
PubMed1982

Solid-phase double-antibody radioimmunoassay of pepsinogen I in serum.

A solid-phase radioimmunoassay for the determination of pepsinogen I in serum has been developed. The antibody was raised in rabbits with pepsinogen I isolated from urine as previously described. Radioiodination was carried out with a chloramine-T procedure resulting in a tracer with excellent shelf life. In the standard procedure with a 24-h incubation time, followed by 2-h incubation with a second antibody coupled to a solid phase, 50 microliter serum was analyzed, standard range 1.88-60 ng PG I. An eight times more sensitive method was also developed using sequential saturation techniques. Specificity studies demonstrated 0.6% crossreactivity with PG II. The immunoreactivity of PG I purified from urine was nearly identical with the immunoreactivity of PG I purified from gastric mucosa. The levels of PG I in serum from 121 control subjects were similar to those obtained with conventional phase separation methods. It is concluded that the method is simple, precise and free from non-specific serum interference.

Animals↗
PubMed1983

Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay.

Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48 000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with alpha-naphthyl phosphate at 30 degrees C and para-nitrophenyl phosphate at 37 degrees C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 micrograms of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9-7.7% with serum at 1.3 to 5.6 micrograms PAP/l.

Acid Phosphatase↗
PubMed1983

Radioimmunoassay of somatostatin in human plasma.

A radioimmunoassay procedure for determination of somatostatin immunoactivity in human plasma is described. Labelling of tyrosine-1-somatostatin is performed by chloramine T. Specific activity of the tracer is 1.34 mol iodine for 1 mol of somatostatin. The immunoreactivity is 85%. Antisera are produced in rabbits at a titre of 179 X 10(-12) mol somatostatin per ml antiserum. Human plasma is extracted by n-butanol/2 mol/l acetic acid. Recovery rate is 85-105%. The intra-assay CV varies from 12% to 1.7%, the inter-assay CV from 16% to 7.5%, depending on the concentration of somatostatin.

Animals↗
PubMed1983

Preparation and metabolism of 125I-sulfobromophthalein.

Metabolism of 125I-sulfobromophthalein (BSP) prepared by the chloramine-T method was studied in rats. 125I-BSP is removed rapidly from the circulation. However, as compared to BSP, its plasma clearance and biliary excretion are delayed, and its accumulation in the liver is prolonged. Although BSP and 125I-BSP show similar binding to albumin in serum, their binding properties to liver cytosolic proteins and to the liver cell plasma membrane organic anion binding protein (OABP) differ. In contrast to the X-, Y- and Z-protein binding of BSP, 125I-BSP binds predominantly to a high molecular weight protein and only a small proportion of 125I-BSP binds to OABP.

Animals↗
PubMed1984

Development and validation of a specific radioimmunoassay for PHI in plasma.

A sensitive and specific radioimmunoassay for the recently isolated neuropeptide with N-terminal histidine and C-terminal isoleucine amide (PHI) has been developed which can detect 3.0 pmol/l of the peptide in plasma. The routine antiserum produced in rabbit had a titre of 1:800 000 and recognized the sequence 3-8 of the 27 amino acid peptide. Labelling of PHI with 125I was performed by the chloramine T method, and labelled PHI was separated from unlabelled on an octadecylsilyl (ODC)-silica column. Non-specific interference in the assay was excluded by extraction of plasma with ethanol to a mean recovery of 82.4%. Plasma samples diluted parallel to the standard curve and behaved as PHI on ODC-silica column. The intra-assay and inter-assay coefficient of variation (CV) values at a level of 24.0 pmol/l were 6.3% and 13.1%, respectively. In 75 normal adults, the fasting PHI concentration ranged from 3.5-30.0 pmol/l with a mean of 14.2 pmol/l. In 235 children, the PHI concentration varied with age. Ingestion of a meal caused a rapid and short-lived increase in the PHI concentration.

Adolescent↗
PubMed1984

Radioimmunoassay of secretin in human serum.

A sensitive radioimmunoassay for secretin has been developed. Antisera were raised against synthetic porcine secretin coupled to bovine serum albumin. N-alpha- desaminotyrosyl -beta-alanyl secretin was radioiodinated by a slight modification of the chloramine-T method. Pure synthetic porcine secretin was used as a standard. Free and bound hormone were separated by dextran-coated charcoal. No cross-reactivity was found with structurally and physiologically related peptides. The sensitivity of the assay was high enough to measure fasting secretin levels in human serum. Patients with acute or chronic pancreatitis had mean serum secretin concentration not significantly different from healthy subjects. In patients with pancreatic carcinoma the mean serum secretin concentration was significantly lower than in healthy subjects, although a wide overlap of the two groups was evident.

Adolescent↗
PubMed1984

Evaluation of a sensitive and specific radioimmunoassay for pancreatic glucagon in human plasma and its clinical application.

A glucagon radioimmunoassay employing antiserum specific for pancreatic glucagon is described. Glucagon was radioiodinated by the chloramine T technique and purified on QAE-Sephadex A 25 to a specific activity of 225 mu Ci/microgram. The standard curve allowed measurements from 12 to 500 pg/ml with sensitivity of 17.5 pg/ml, precision of 6.3-14.9% (CV, within-assay) and 5.6-10.7% (CV, between-assay). Recovery was between 82 and 112%. Fasting plasma glucagon levels in diabetics, obese subjects, acromegalics and patients with Cushing's syndrome were greater than in normals (22.0 +/- 91 pg/ml; mean +/- SD). Very low glucagon levels after oral glucose suppression (15.2 +/- 3.1 pg/ml) in normals and greatly increased values after arginine in insulin-dependent diabetics (271.0 +/- 132.3 pg/ml) could be determined.

Acromegaly↗
PubMed1985

Heterogeneity of 125I-labeled human thyroglobulin preparations.

This study was undertaken to evaluate the heterogeneity and stability of 125I-labeled human thyroglobulin (Tg) tracers. Tg, labeled with 125I by either a Chloramine T (CT) or a Glucose Oxidase/Lactoperoxidase (GO) method, showed considerable heterogeneity of labeled components, the relative proportions of which were a function of the Tg preparation and the iodination method used. The four largest components had apparent molecular weights as follows: A = 1 200 000 Da; B = 670 000 Da; C = 530 000 Da and D = 290 000 Da. Both B and C were immunoactive. B was considered to be 19S Tg. A non-specific binding difference, (NSB delta) between nonhuman matrices used for diluting standards and human sera was found for the partly immunoactive aggregate component A, (5-20%) and the nonimmunoactive component D, (20-50%), but was minimally present for components B and C (less than 5%). The [125I]19S Tg(B), stored at -70 degrees C, showed rapid spontaneous decomposition with time (50% lost by 8 days), with generation of C, D and iodide. The loss of B was related to specific activity and was least in GO labels. 125I labeling of Tg by GO produced tracers with better immunoactivity, stability and lower NSB delta than comparative CT tracers. Definitive purification and repurification of [125I]Tg tracers before use is necessary in order to remove degradation products with the potential to compromise the accuracy and specificity of serum Tg radioimmunoassay (RIA) measurement.

Chromatography↗