scholarly journals Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

1970 ◽  
Vol 120 (4) ◽  
pp. 831-836 ◽  
Author(s):  
J. Méšter ◽  
D. M. Robertson ◽  
Patricia Feherty ◽  
A. E. Kellie
Keyword(s):  
Dissociation Constant ◽  
Oestrogen Receptor ◽  
Scatchard Plot ◽  
Assay Method ◽  
Physiological Conditions ◽  
High Affinity ◽  
Receptor Sites ◽  
Assay Procedure ◽  
Order Of Magnitude

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k−1) and low-affinity (k−2) receptor sites and [3H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.

Radioimmunoassay for 4-hydroxyoestrone in human urine

1981 ◽  
Vol 97 (2) ◽  
pp. 251-257 ◽  
Author(s):  
Günter Emons ◽  
Claudia Mente ◽  
Rudolf Knuppen ◽  
Peter Ball
Keyword(s):  
Human Urine ◽  
Gas Chromatography Mass Spectrometry ◽  
Clear Cut ◽  
High Affinity ◽  
Assay Procedure ◽  
Female Children ◽  
Bovine Serum Albumin Conjugate ◽  
Order Of Magnitude ◽  
Excretion Rates

Abstract. Under the protection of ascorbic acid a 4-hydroxyoestrone-bovine serum albumin conjugate was prepared containing intact 4-hydroxyoestrone as determined by gas chromatography-mass spectrometry. Using this antigen, antibodies with high affinity and specificity for 4-hydroxyoestrone were raised in rabbits. An assay procedure for the determination of 4-hydroxyoestrone in human urine and the assessment of its reliability are described. The following urinary excretion rates were found: male children 0.29 μg/24 h, female children 0.35 μg/24 h, men (20–40 years) 1.6 μg/24 h, men (>50 years) 1.8 μg/24 h, women, follic. 2.0 μg/24 h, pre-ov. 5.3 μg/24 h, luteal 2.4 μg/24 h, women, pregnant, first trim. 30.0 μg/24 h, second trim. 64.0 μg/24 h, third trim. 48.0 μg/24 h, women, post-men. 1.5 μg/24 h. Thus the amounts of 4-hydroxyoestrone excreted in human urine are about 1/3 to 1/10 of those of 2-hydroxyoestrone. During the menstrual cycle the excretion rates of 4-hydroxyoestrone are in the same order of magnitude as those of oestradiol and show a clear-cut pre-ovulatory peak.

DETERMINATION OF SOLUBLE FIBRIN IN PLASMA WITH A CHR0M0GENIC KIT METHOD UTILIZING THE HIGH AFFINITY PLASMIN SUBSTRATE S-2390

1987 ◽  
Author(s):  
E Ersdel ◽  
M Andersson ◽  
S Rosen
Keyword(s):  
Reagent Blank ◽  
Soluble Fibrin ◽  
Standard Curve ◽  
High Affinity ◽  
Absorbance Unit ◽  
Assay Procedure ◽  
Bovine Plasma ◽  
Fresh Frozen ◽  
Reliable Differentiation

A sensitive and quantitative assay of soluble fibrin is of clinically diagnostic relevance in an early thrombotic state where there is a risk for development of DIC. Recently Wiman and Ranby (Thromb. Haemostas 55, 189-193 (1986)) published a spectro-photometric assay which met these criterions. The single-stage assay procedure is based upon activation of Glu-Plasminogen to Plasmin by t-PA in the presence of soluble fibrin and hydrolysis of the chromogenic plasmin substrate S-2390, H-D-Val-Phe-Lys-pNA, which has a high affinity for plasmin. The rate of plasmin generation is correlated to the amount of soluble fibrin monomers present in the sample.A complete kit containing optimized, stable reagents has now been developed which allows a quantitative determination of soluble fibrin in the range 30-200 nmol/1 within 30 min. at room temperature (20-25°C). The assay procedure is straightforward involving addition of 200 pi diluted plasma sample to 200 pi Glu-Plasminogen and 100 ul of a t-PA/S-2390-reagent.The results show a high resolution of the standard curve as illustrated by a AA405 amounting to about one absorbance unit between a 200 nmol/1 sample of soluble fibrin and the reagent blank, some variation, ±0.1 absorbance unit, being caused mainly by differences in temperature. In combination with an intra-assay variation coefficient = 6.3% and 5.0% at 150 and 50 nmol/1, respectively, this will allow safe and reliable differentiation of pathological levels of soluble fibrin from levels found in healthy subjects (below 10 nmol/1). A similar precision is also obtained when the assay is performed in microplates.In the original procedure fresh frozen human plasma was utilized as a dilution medium for soluble fibrin. Comparisons with carefully collected bovine plasma proved this source to be a convenient substitute. Furthermore, lyophilization of the bovine plasma did not produce any significant degradation of fibrinogen which otherwise might interfere in the assay. This simple kit procedure should make it a suitable tool in early determinations of soluble fibrin in a number of pathological states which may result in severe haemostatic disturbances.

A HIGH-AFFINITY BINDING SITE FOR THE ANTIOESTROGENS, TAMOXIFEN AND CI 628, IN IMMATURE RAT UTERINE CYTOSOL WHICH IS DISTINCT FROM THE OESTROGEN RECEPTOR

1981 ◽  
Vol 91 (1) ◽  
pp. 155-161 ◽  
Author(s):  
L. C. MURPHY ◽  
R. L. SUTHERLAND
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Dissociation Constants ◽  
Significant Proportion ◽  
Immature Rat ◽  
High Affinity ◽  
Saturable Binding ◽  
Receptor Sites

A high-affinity, saturable antioestrogen binding site, which does not bind oestradiol, has been reported to exist in a number of oestrogen target tissues but not in the immature rat uterus. This study reports the results of a more thorough search for this site in immature rat uterine cytosol. When concentrations of uterine cytoplasmic oestrogen receptor were selectively depleted by translocation of 90–95% of the cytoplasmic oestrogen receptor to the nucleus, saturation analysis studies revealed that the antioestrogens, tamoxifen and CI 628, were bound to high-affinity, saturable binding sites which were present at about 2·5 times the concentration of the residual oestrogen receptor sites. Oestradiol could only partially inhibit the binding of tritiated antioestrogens to their saturable binding sites in this material indicating that a significant proportion of these sites were distinct from the oestrogen receptor sites. This was confirmed in experiments where oestrogen receptor sites were saturated in vitro with oestradiol and high-affinity, saturable sites for CI 628 and tamoxifen were still present. The CI 628 and tamoxifen had high affinity for these sites with dissociation constants of 1·0–1·6 nmol/l. These specific antioestrogen binding sites were present at about 5% of the concentration of oestrogen receptors in normal immature rat uterine cytosol which probably explains their previous lack of detection in this material.

Clinical studies of the use of a fluorogenic substrate assay method for the determination of plasminogen

1979 ◽  
Author(s):  
Douglas Triplett ◽  
Cathy Harms ◽  
Lori Hermelin ◽  
Rolf Huseby ◽  
Gary Mitchell ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Assay Method ◽  
Fluorogenic Substrate ◽  
Normal Range ◽  
Thrombosis Research ◽  
Normal Ranges ◽  
Assay Procedure ◽  
Medical Population ◽  
Pulmonary Bypass

Utilization of a recently reported method for the determination of plasminogen by fluorometric technique (Ref. 3, Pochron, et. al., Thrombosis Research, Vol. 13, pg. 733, 1978) was compared with acceptable methodologies (e.g. the caseinolytic and radial–immunodiffusion assays). Excellent results were seen with the normal range of 2.4–3.8 CTA U/ml of plasminogen by the synthetic substrate assay as compared to 8.7–14.3 mg/dl by the radialimmunodiffusion assay. Normal ranges for the caseinolvtic assay were seen to be 2.2–4.0 CTA U/ml. Patients’ studies were performed on a general medical population but centered primarily on “pre” and “post” operative coronary-pulmonary bypass patients. In all cases studied, a drop in Plasminogen level was noted in this group of patients following surgery (20-50%). Other clinical data will be presented indicating that the determination of plasminogen by the fluorometric assay procedure is sensitive and accurate for clinical situations.

Clinical Studies of the Use of a Fluorogenic Substrate Assay Method for the Determination of Plasminogen

1979 ◽  
Author(s):  
Douglas A. Triplett ◽  
Cathy Harms ◽  
Lori Hermelin ◽  
Rolf M. Huseby ◽  
Gary A. Mitchell ◽  
...  
Keyword(s):  
Radial Immunodiffusion ◽  
Assay Method ◽  
Fluorogenic Substrate ◽  
Normal Range ◽  
Thrombosis Research ◽  
Normal Ranges ◽  
Assay Procedure ◽  
Medical Population ◽  
Pulmonary Bypass

Utilization of a recently reported method for the determination of plasminogen by fluorometrit technique (Ref. 3, Pochron, et. al., Thrombosis Research, Vol. 13, pg. 733, 1978) was compared with acceptable methodologies (e.g. the caseinolvtic and radial-immunodiffusion assays). Excellent results were seen with the normal range of 2.4–3.8 CTA U/ml of plasminogen by the synthetic substrate assay as compared to 8.7-14.3 mg/dl by the radial immunodiffusion assay. Normal ranges for the caseinolvtic assay were seen to be 2.2-4.0 CTA U/ml. Patients’ studies were performed on a general medical population but centered primarily on “pre” and “post” operative coronarv-pulmonary bypass patients. In all cases studied, a drop in Plasminogen level was noted in this group of patients following surgery (20–50%). Other clinical data will be presented indicating that the determination of plasminogen by the fluorometric assay procedure is sensitive and accurate for clinical situations.

Evaluation of an Automated Dual-Channel Hydroxylamine Assay Procedure for Formulated Penicillin Products. II. Intact Solid Dosage Formulations

1974 ◽  
Vol 57 (6) ◽  
pp. 1325-1337
Author(s):  
Jonathan R Lane
Keyword(s):  
Drug Administration ◽  
Automated System ◽  
Assay Method ◽  
Manual Method ◽  
Solid Dosage ◽  
Dual Channel ◽  
Assay Procedure ◽  
Wide Range ◽  
Ampicillin Trihydrate

Abstract The dual-channel hydroxylamine method of Stevenson, Bechtel, and Coursen for the potency determination of formulated penicillins has been evaluated for use as an official Food and Drug Administration (FDA) alternative assay method for solid dosage formulations. A modified Technicon SOLIDprep Sampler I was used to assay a wide range of penicillin capsules and compressed tablets. In some instances, as many as 10 tablets/sample cup were tested. Results by this method and the official FDA manual method are compared. Some of the problems encountered in the analysis of ampicillin trihydrate capsule formulations and with compressed, film-coated tablets are also described. Based on the performance of the automated system and the data obtained, recommendations have been made to make the dual-channel hydroxylamine procedure an official FDA alternative assay technique for penicillin formulations.

Comparison of Chemical and Microbiological Methods for the Determination of Procaine Penicillin in Premixes and Mixed Feeds

1963 ◽  
Vol 46 (3) ◽  
pp. 429-433
Author(s):  
Stanley E Katz
Keyword(s):  
Chemical Method ◽  
Microbiological Assay ◽  
Assay Method ◽  
Microbiological Method ◽  
Procaine Penicillin ◽  
Assay Procedure ◽  
Microbiological Methods ◽  
Mixed Feeds

Abstract A chemical and a microbiological method of analysis for procaine penicillin in premixes and mixed feeds have been compared. The microbiological assay method was a typical cylinderplate assay procedure. The chemical method was based upon the conversion by base of penicillins to penicilloic acids. In the eight premixes studied, the chemical-to-microbiological ratio of results varied from 0.92 to 1.17. In four mixed feeds, the ratio varied from 0.97 to 1.09. In general, the chemical method yielded slightly higher results than the microbiological method. There was little difference between methods in regard to reproducibility and accuracy.

scholarly journals QUANTITATION OF TISSUE-BOUND RENAL AMINOPEPTIDASE BY A MICRODENSITOMETRIC TECHNIQUE

1966 ◽  
Vol 14 (1) ◽  
pp. 53-63 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Tissue Section ◽  
Diazonium Salt ◽  
Rat Kidney ◽  
Significant Proportion ◽  
Enzymic Activity ◽  
Assay Method ◽  
Assay Procedure ◽  
Order Of Magnitude ◽  
Proximal Tubuli ◽  
Ph Level

Relationships between biochemical and histochemical assay systems can be evaluated by appropriate techniques. With rat kidney as the test object the enzyme localized in the tissue section hydrolyzing l-leucyl-β-naphthylamide (LNA) was found to be identical with a purified particle-bound, cobalt-activated aminopolypeptidase previously characterized biochemically. A soluble sulfhydryl-dependent aminopeptidase, present in the rat kidney and also known to hydrolyze LNA, was not demonstrated in the histochemical system, although the diazonium salt employed incidentally caused the insolubilization in the tissue section of a significant proportion of previously extractable, particle-bound enzyme. Linearity between the enzymic activity present and the measurable enzymic activity was demonstrated to exist at the substrate concentration, pH level, section thickness and diazonium salt concentrations used in the assay procedure, but did not exist when these factors were varied beyond certain well defined limits. In addition, the effect of diazonium salt on both the inhibition and insolubilization of enzyme in the tissue section could be quantitated. Based on this evaluation of the enzymes hydrolyzing LNA in the rat kidney, a microdensitometric assay method employing a constant flow incubating chamber was developed to characterize and quantitate the LNA-hydrolyzing enzyme in the proximal tubuli. This study defines many of the parameters necessary for the future investigation by quantitative and qualitative methods of histochemical systems capable of providing resolutions of a higher order of magnitude and the retention of a greater proportion of extractable enzyme within the tissue section.

scholarly journals Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, pregnancy, maturation and after ovariectomy

1970 ◽  
Vol 120 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Patricia Feherty ◽  
D. M. Robertson ◽  
H. B. Waynforth ◽  
A. E. Kellie
Keyword(s):  
Early Pregnancy ◽  
Similar Pattern ◽  
Quantitative Method ◽  
Oestrous Cycle ◽  
High Concentration ◽  
Physiological Conditions ◽  
High Affinity ◽  
Receptor Sites ◽  
Receptor Concentration ◽  
Cyclic Changes

A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.

The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

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