Determination Of Aldosterone In Urine Research Articles

Removal of contaminating substances before gas-liquid-chromatographic determination of aldosterone in urine.

This paper deals with removal of contaminants before gas-chromatographic determination of aldosterone in urine. Urine is incubated with beta-glucuronidase, which hydrolyzes all beta-glucuronides except aldosterone-18-glucuronide. The contaminants (the aglycones released and other methylene chloride-soluble substances) are extracted with methylene chloride. Solvolysis of the aqueous phase liberates aldosterone from aldosterone-18-glucuronide, which then is extracted with methylene chloride and oxidized by use of periodic acid. The resulting lactone can be easily separated by one-dimensional thin-layer chromatography and determined by gas-liquid chromatography.

Die bestimmung von aldosteron in harn und plasma

Determination of aldosterone in urine and plasma The method described permits an exact and rapid determination of aldosterone in urine and plasma. The reliability of the method is based on the separation of aldosterone from contaminating steroids by thin-layer chromatography. The mobile phase used was: cyclohexane—ethyl acetate (20:80). The steroids were extracted by dichloromethane. Plasma was extracted directly, and urine after hydrolysis with sulfuric acid (pH = 1). Recovery before radioimmunological analysis of aldosterone was 54.8 ± 7.2 (S.D.) % ( n=40) for urine samples, and 39.1 ± 4.4 % ( n=60) for plasma samples. The coefficient of variation for multiple determinations of aldosterone was for urine 8.2% for low ( n = 10) and 14.5% for high ( n=10) values; for plasma the respective values were 18.9% ( n=10) and 7.8% ( n=10). The sensitivity of the determination of aldosterone was for urine 0.04 μg per 24-h volume ( n=10) and for plasma 4.4 ng per 100 ml ( n=10). The method avoids pitfalls due to the cross-reaction of anti-aldosterone serum with other materials.

Simple fluorometric determination of aldosterone in urine without use of isotopes or chromatography.

Aldosterone 18-glucuronide in urine is hydrolyzed by adjusting the pH to 1.0 and allowing the mixture to stand overnight at room temperature. The free aldosterone is then extracted into dichloromethane, which is washed with carbonate to remove acidic compounds and evaporated. The residue is partitioned between a nonpolar organic phase and an aqueous phase, and the aldosterone oxidized to the 13-carboxylic acid derivative with Benedict's qualitative glucose reagent. Neutral compounds are extracted from this oxidation mixture with dichloromethane at pH 7.5, the mixture is acidified, and the oxidized aldosterone extracted into dichloromethane. After washing with pH 3.5 buffer, this extract is evaporated and the oxidized aldosterone determined fluorometrically via a two-stage reaction with sulfuric acid/water (85/15 by vol) and methanol containing ferric chloride.

An improved method for the gas-liquid chromatographic determination of aldosterone in urine

An improved method for the gas-liquid Chromatographic determination of aldosterone in urine has been developed. The method involves the following steps: (1) Hydrolysis of the aldosterone conjugate (pH 1-labile metabolite) by cold hydrochloric acid. (2) Addition of [1,2- 3H 2]aldosterone as a tracer. (3) Purification of aldosterone by adsorption chromatography on Amberlite XAD-2, followed by distribution between solvents and thin-layer chromatography on silica gel. (4) Oxidation of aldosterone by periodic acid to aldosterone-γ-lactone. (5) Purification of the γ-lactone by thin-layer chromatography. (6) Gas-liquid chromatography of the purified sample by using a solid injection system and a flame ionisation detector. (7) Measurement of radioactivity to correct for losses during the working-up procedures. By using a time-controlled automatic solid injection system, the practicability of the method is considerably enhanced. At the same time, the sensitivity of the method is improved, since larger portions of the samples can be utilised for gas chromatographic determination as compared to the liquid injection system.

Measurement of urinary aldosterone by a simplified radioimmunoassay procedure

A relatively simple and rapid radioimmunoassay for the determination of aldosterone in urine has been developed, employing tritiated aldosterone and a commercially available antiserum of high titer. Chromatography of specimens prior to immunoassay has been found to be unnecessary. The method involves extraction of a 3 ml aliquot of urine with dichloromethane (DCM), followed by a 24-h acid hydrolysis and a second DCM extraction. Extracts are ready for immunoassay after filtration through silica gel. Half-saturation with ammonium sulphate is used to separate free from antibody-bound aldosterone. Recovery is 90%, between-run coefficient of variation is ±14%, blanks are negligible, and comparison with the double isotope derivative method show no statistical difference and the same normal range.

A Rapid and Practical Determination of Aldosterone in Urine by Double Isotope Method

A double isotope derivative method for urinary aldosterone determination was improved in terms of economy (to one fifth the original cost) and efficiency (to less than half the time) by a combination of several individual improvements.Aldosterone-4-14C was used as an indicator, eliminating the procedure of preparing aldosterone-14C-diacetate. 3H-acetylation was done after, instead of before, dichloromethane extraction and a thin layer-chromatography, to avoid unnecessary acetylation of abundant contaminants. The cost of radioisotope was thus reduced to one tenth. dl-Aldosterone diacetate, added as a carrier, was easily located on thin layers by ultraviolet light. Two dimensional thin layer, instead of repeating one dimensional paper chromatography, was employed for the purification, thereby shortening the time to one twentieth to thirtieth, and enabling to eliminate the exidation of the diacetate to the monoacetate. The specific radioactivity of 3H-acetic anhydride was determined by acetylation of dlaldosterone mixed with urine, instead of cortisone or dl-aldosterone, for both economy and calibration. One technician can thus work out with 4-8 samples a week.Aldosterone excretion in 24 hr, thus determined, was 10.99±3.83 (S.D.)μg for normal subjects and 32.12±8.38μg for preoperative primary aldosteronism with a postoperative decrease to 12.03±4.10μg.

RAPID METHOD FOR DETERMINATION OF ALDOSTERONE IN HUMAN URINE BY GLASS PAPER CHROMATOGRAPHY*

A method for the determination of aldosterone in urine is described, which utilizes rapid chromatography on glass fiber paper. After acid hydrolysis, chloroform extracts of the urine are chromatographed on silica-gel columns, run on 2 glass paper chromatographic systems, acetylated, and again separated by 2 additional runs in separate glass paper chromatographic systems. Final determination of aldosterone is performed by measurement of alkaline fluorescence. The specificity and precision of the method, the recovery of steroid by this technic, and the range of values in normal man are presented. The advantages of glass paper chromatography are pointed out, not the least important of which is that each chromatographic step requires only fifteen minutes.

Chemical determination of aldosterone in urine.

ABSTRACT A method for the determination of aldosterone in urine is presented, which includes the following steps: acid hydrolysis and extraction, column chromatography on silica gel, 2 paper chromatographic systems, acetylation, a third paper chromatographic system, and fluorometry directly on the paper using alkaline fluorescence. Data are presented on precision, recovery, duration of acid hydrolysis, acid versus enzymatic hydrolysis, stability of urine samples and normal values.

A new method for the colorimetric determination of aldosterone in urine.

A new method for the colorimetric determination of aldosterone in urine.