PROCEDURE FOR DETERMINATION OF ALDOSTERONE IN HUMAN PERIPHERAL PLASMA BY DOUBLE-ISOTOPE DERIVATIVE ASSAY, AND ITS APPLICATION FOR MEASUREMENT OF SECRETORY RATE AND URINARY EXCRETION

1967 ◽  
Vol 45 (12) ◽  
pp. 1919-1936 ◽  
Author(s):  
Wojciech Nowaczynski ◽  
Jack Silah ◽  
Jacques Genest
Keyword(s):  
Acetic Anhydride ◽  
Urinary Excretion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Plasma Aldosterone ◽  
Chromatographic Purification ◽  
Secretory Rate ◽  
Peripheral Plasma ◽  
Double Isotope

This is a report of a procedure for the determination of peripheral plasma aldosterone, in which 14C-labeled aldosterone serves as the marker, and the acetylating agent is relatively inexpensive tritium-labeled acetic anhydride of high specific activity. A new method for the chromatographic purification of aldosterone and aldosterone diacetate is presented. This procedure provides a better separation of these two compounds from the unlabeled and labeled impurities.

scholarly journals The determination of oestradiol and oestrone in the plasma of the domestic fowl by a method involving the use of labelled derivatives

1968 ◽  
Vol 106 (1) ◽  
pp. 77-86 ◽  
Author(s):  
J. E. O'Grady
Keyword(s):  
Human Plasma ◽  
Paper Chromatography ◽  
Domestic Fowl ◽  
Specific Activity ◽  
High Specific Activity ◽  
Double Labelling ◽  
Plasma Samples ◽  
Labelling Technique ◽  
Preliminary Purification

1. A method involving the use of triple-labelled derivatives has been developed for the determination of total oestrone and oestradiol in the plasma of the domestic fowl. The double-labelling technique devised by Svendsen (1960) for the determination of free oestrogens in human plasma was modified to enable the total oestrogen recovery to be determined for each sample. 2. [6,7−3H2]Oestradiol-17β is added to the plasma samples (1–10ml.), which are hydrolysed with acid and the phenolic steroids then extracted and partially purified. The extract is esterified with iodobenzene-p[35S]-sulphonyl chloride of high specific activity. After addition of standard oestrogen [131I]iodobenzene-p-sulphonates the esters are finally purified by paper chromatography. 3. The oestrogens are determined by comparing the 3H/35S and 131I/35S ratios in the purified esters with similar ratios of appropriate standards. 4. With this procedure the recoveries of oestrone and oestradiol after hydrolysis were 70–85% and 72–84% respectively, and after hydrolysis and preliminary purification 38–53% and 39–51% respectively. With this procedure up to 500ng. of oestradiol can be determined. The sensitivity of the technique for oestrone is 3·0ng. and for oestradiol 2·1ng. 5. The ranges of oestradiol and oestrone concentrations found in six plasma samples were 8·3–21·4ng./ml. and 15·2–31·6ng./ml. respectively.

Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten

1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf
Keyword(s):  
Quantitative Determination ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ether Extract ◽  
Qualitative And Quantitative ◽  
Work Up ◽  
First Time ◽  
Very High ◽  
Tenebrio Molitor Larvae

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

The determination of precursors to urinary methyl-malonic acid in man; measurement of urinary excretion and specific activity

1969 ◽  
Vol 23 (2) ◽  
pp. 279-287 ◽  
Author(s):  
Joseph A. Degrazia ◽  
Mathews B. Fish ◽  
Myron Pollycove ◽  
Michael Ponnamperuma
Keyword(s):  
Urinary Excretion ◽  
Malonic Acid ◽  
Specific Activity

Improvements in the determination of the turnover rate of plasma thyroxine in avian species: application to the Japanese quail

1987 ◽  
Vol 114 (2) ◽  
pp. 191-198 ◽  
Author(s):  
E. J. Cookson ◽  
J. Glover
Keyword(s):  
Japanese Quail ◽  
Half Life ◽  
Turnover Rate ◽  
Specific Activity ◽  
Simple Procedure ◽  
High Specific Activity ◽  
Future Application ◽  
Sodium Thiocyanate ◽  
Plasma Samples

ABSTRACT The disappearance of the thyroid hormone thyroxine (T4) from plasma in fully grown male Japanese quail can be described as a first order process with a rate constant of 0·178 ± 0·013/h (mean±s.e.m., n = 8), which represents a half-life of 3·90 h. A small amount of [125I]T4 in relation to total circulating T4 was injected i.v. into Japanese quail and plasma samples were taken at appropriate time-intervals for the determination of residual plasma radioactivity. The rate of disappearance of [125I]T4 was subsequently equated to the turnover rate of the endogenous hormone. Previous methods were modified to overcome problems arising from possible disturbance of plasma T4 metabolism, recirculation of radiolabelled iodide, and to purify the [125I]T4 from the plasma samples. By using labelled T4 of very high specific activity, the amount of [125I]T4 administered was kept much smaller than has been used in previous studies on Japanese quail, thus limiting any interference with plasma T4 dynamics. To minimize any disturbance of plasma T4 metabolism, only four blood samples were taken, at three-hourly intervals after the injection of [125I]T4. The rapid turnover of T4 produced a large amount of labelled inorganic iodide, the re-entry of which into the plasma T4 pool was inhibited by s.c. administration of sodium thiocyanate 1 h before injection of[125I]T4. Assay of the true [125I]T4 turnover was significantly improved over that used in previous studies by purifying the [125I]T4 from the plasma samples chromatographically. The samples were applied to small Sephadex G-25 columns with sodium hydroxide (0·1 mol/l) as the eluant. This simple procedure clearly separated the [125I]T4 from the other radioiodinated plasma components such as free iodide, non-hormonal iodinated proteins and triiodothyronine (T3), thus enabling a more accurate assessment of the residual labelled T4 concentration in the plasma and hence the T4 half-life. The future application of this method to the study of plasma T4 turnover under various experimental conditions is discussed and the possible involvement of T4 turnover studies in the assessment of T4 to T3 conversion is considered. J. Endocr. (1987) 114, 191–198

A DOUBLE ISOTOPE DERIVATIVE METHOD FOR THE DETERMINATION OF ALDOSTERONE IN PERIPHERAL PLASMA

1961 ◽  
Vol 37 (4) ◽  
pp. 541-558 ◽  
Author(s):  
Ejgil Bojesen ◽  
Hans Degn
Keyword(s):  
Isotope Dilution ◽  
Half Life ◽  
Acid Anhydride ◽  
Hydroxyl Group ◽  
Aldosterone Level ◽  
Peripheral Plasma ◽  
Derivative Method ◽  
Double Isotope

ABSTRACT The details of a method for the determination of aldosterone at the level of 1 ng or more are described. A combination of the isotope derivative principle and the isotope dilution principle, using 35S and 131I labelled piodobenzenesulfonic acid anhydride (pipsan) as the labelled reagents, has been employed. A quantitative or almost quantitative and reproducible esterification of the 21-hydroxyl group of aldosterone and an 80% recovery of the steroid added to plasma can be achieved by the procedure described. The blank value has been determined by the analysis of epripheral plasma of adrenalectomized dogs and was found to be indistinguishable from zero and therefore less than 3 ng per 100 ml of plasma. In salt depleted anaesthetized dogs the aldosterone level was 20–30 ng per 100 ml. The concentration increased considerably during adrenalectomy and decreased with a »half life« of 20–30 minutes from the time this was completed.

THE DEVELOPMENT OF A RADIOIMMUNOASSAY FOR OXYTOCIN: RADIO-IODINATION, ANTIBODY PRODUCTION AND SEPARATION TECHNIQUES

1970 ◽  
Vol 46 (2) ◽  
pp. 269-278 ◽  
Author(s):  
T. CHARD ◽  
M. J. KITAU ◽  
J. LANDON
Keyword(s):  
Lymph Node ◽  
Human Plasma ◽  
Rapid Method ◽  
Ammonium Sulphate ◽  
Antibody Production ◽  
Specific Activity ◽  
High Specific Activity ◽  
Separation Techniques ◽  
Ammonium Sulphate Precipitation

SUMMARY A simple and rapid method is described for labelling oxytocin with 131I at a high specific activity. This method is compared with those of previous workers. A satisfactory antiserum has been raised by direct intra-lymph node injection of oxytocin adsorbed to carbon microparticles. A number of methods for separating antibody-bound from free oxytocin are described, and reasons given for preferring a procedure using ammonium sulphate precipitation. These data form the basis for developing a radioimmunoassay intended for the determination of oxytocin in human plasma.

ULTRAFILTRATION STUDIES OF STEROID-PROTEIN BINDING

1961 ◽  
Vol 23 (2) ◽  
pp. 129-137 ◽  
Author(s):  
P. S. CHEN ◽  
I. H. MILLS ◽  
F. C. BARTTER
Keyword(s):  
Protein Binding ◽  
Specific Activity ◽  
Binding Protein ◽  
High Specific Activity ◽  
Wide Range

SUMMARY A method is described for determination of the extent of protein binding of steroids by ultrafiltration after addition of radioactively-labelled steroid tracers of high specific activity. Results obtained with specifically labelled 14C-steroids did not differ significantly from those with steroids randomly labelled with tritium. A number of steroids have been studied with this technique. The 'S' shaped curve obtained with some corticosteroids by plotting percentage ultrafilterable against steroid added to the plasma confirmed the presence of the corticosteroid-binding protein which has greater affinity for steroid than albumin. This method of plotting the ultrafiltration data afforded a method of assessing the amount of binding protein (or the number of sites). Cortisone, 17-hydroxy-11-deoxycorticosterone, cortisol, and Δ1 cortisol showed this type of curve. Binding of aldosterone, progesterone and 17-ketosteroids occurred to the same extent in plasma as in 5% albumin. The ultrafilterable fraction of these steroids in plasma was constant over a wide range of total steroid concentration. Binding of testosterone was greater with plasma than with albumin, and did not decrease upon addition of large amounts of testosterone.

PLASMA ALDOSTERONE DETERMINATION WITH 35S-p-TOLUENE-SULPHONIC ANHYDRIDE

1972 ◽  
Vol 70 (3) ◽  
pp. 552-566 ◽  
Author(s):  
D. Scholer ◽  
A. M. Riondel ◽  
E. L. Manning
Keyword(s):  
Plasma Level ◽  
Thin Layer ◽  
Water Samples ◽  
Regression Line ◽  
Mean Value ◽  
Plasma Aldosterone ◽  
Peripheral Plasma ◽  
Derivative Method ◽  
Replicate Measurements ◽  
Double Isotope

ABSTRACT The double isotope derivative method for plasma aldosterone, (Bojesen & Thuneberg 1967), has been applied to human peripheral plasma. 35S-p-toluene-sulphonic anhydride was used as the labelled reagent (100–150 mCi/mEq.) and 3H-aldosterone (30 Ci/mm; 0.05 ng added to each sample) as indicator. The initial derivative (aldosterone-21-35S-tosylester) was transformed to its 11,18-γ-lactone and finally to its 3-dinitrophenylhydrazone. Purification was achieved by 1 paper chromatography and 4 thin-layer chromatographies. The overall-recovery was 10 ± 2.7 (sd)%, n = 134. The non-specific blank was 0.053 ± 0.044 (sd), when determined on water samples, and 0.068 ± 0.034 (sd) ng/5 ml, when determined on adrenalectomised human plasmas (non-specific water blank of the same series: 0.064±0.037 (sd) ng). The addition of a known amount of aldosterone to a plasma resulted in a linear relationship over the range examined: y = (0.947 ± 0.013) × + 0.696, P = 0.000, and aldosterone from 1.0 to 5.0 ng was measured with an accuracy of 95%. Replicate measurements of different volumes of the same plasma from 1.0 to 7.5 ml showed a calculated regression line of y = (0.148 ± 0.005) × −0.01, P = 0.002. Replicate measurements of the same plasma (n = 8) gave a mean value of 0.14±0.01 (sd) ng, a range between 0.13 and 0.15 ng and a coefficient of variation of 7%. Thus, it was possible to determine a peripheral plasma level of 7 ng/100 ml in 2 ml plasma and a level of 3.5 ng/100 ml in 4 ml plasma.

RADIOIMMUNOASSAY OF [8-ARGININE]-VASOPRESSIN

1975 ◽  
Vol 80 (3) ◽  
pp. 444-452 ◽  
Author(s):  
P. Czernichow ◽  
U. Merkelbach ◽  
M. B. Vallotton
Keyword(s):  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Association Constant ◽  
Specific Activity ◽  
Limit Of Detection ◽  
High Specific Activity ◽  
High Association ◽  
Theoretical Maximum

ABSTRACT A radioimmunological method for the determination of the human antidiuretic hormone, [8-arginine]-vasopressin (AVP), is described in detail. The antiserum has been raised in rabbits injected with AVP adsorbed onto charcoal particles and is used at a final dilution of 1:200 000. It contains antibodies directed specifically against the C-terminal tripeptide and possesses a high association constant of 8.2× 1012 1/mole. AVP is labelled in monoiodinated form (as proven after pronase digestion) at a high specific activity close to the theoretical maximum after purification from unlabelled hormone on Sephadex gel. The standard curves are characterized by a Bo of 55 %, a limit of detection ascertained at least at 3 pg (10 % displacement) and a 50 % displacement achieved with 35.5 pg. The index of precision λ ranges from 0.033 to 0.042. The conditions of the assay (buffer's composition and pH, timing) were systematically varied and tested. In addition the result of immunization of chicken with AVP is reported. The assay is adequate for the measurement of AVP in urine and plasma and will be described in forthcoming papers.

The use of p-Iodophenyl[125I] isothiocyanate for determination of N-terminal amino acids in nanomole quantities of protein

1975 ◽  
Vol 28 (10) ◽  
pp. 2289 ◽  
Author(s):  
CJ Burrell ◽  
PD Cooper ◽  
JM Swan
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coupling Reaction ◽  
Molar Content ◽  
Terminal Residue ◽  
Terminal Amino

Identification of the N-terminal residue of 2.5-5 nmol of protein was possible by means of a modified Edman reaction and p-iodophenyl[125I] isothiocyanate of high specific activity. The second and third residues could also be identified. Yields of the terminal residue were 25-50%, allowing approximate quantitation of the molar content of protein. Use of the isotope allowed certain steps of the degradative cycle to be examined in some detail, and preparation of this relatively non- volatile labelled Edman reagent was safe, cheap and convenient. ��� The synthesis and properties of the p-iodophenylthiohydantoins of 19 amino acids, and some side products of the coupling reaction, are described.

Sign in / Sign up

Export Citation Format

Share Document