临床化学分析方法：Aldosterone

Kaplan: Clinical Chemistry, 5th Edition

Clinical References - Methods of Analysis

Aldosterone

Hassan M.E. Azzazy i

Name:Aldosterone

Clinical significance:click here

Molecular mass: 360.4 D

Chemical class: Mineralocorticoid

Refer to Chapter 51, Adrenal Hormones and Hypertension, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.

Students’ Quick Hyperlink Review

•Principles and analysis and current usage

•Reference and preferred methods

•Specimen

•Interferences

•Aldosterone reference intervals

•Interpretation

•Aldosterone performance goals

•References

•Aldosterone methods summary table

Principles of Analysis and Current Usage

Aldosterone is the most important human mineralocorticoid; it is secreted by the zona glomerulosa of the adrenal cortex and stimulates sodium transport across cell membranes in the distal renal tubules. Aldosterone plays a major role in homeostasis of sodium and potassium and maintenance of arterial blood pressure in the sodium-depleted state. The renin-angiotensin system regulates aldosterone secretion. Low sodium concentration or low blood volume causes release of renin from kidney cells, which mediates release of aldosterone.

Aldosterone exists in picomolar concentrations in serum, and thus sensitive assays are necessary for its measurement. Several methods are available for determination of aldosterone in blood and urine (Table 1). These include immunoassays, isotope-dilution gas chromatography/mass spectrometry ID-(GC/MS) [1], high-performance liquid chromatography-tandem mass

i Aldosterone

New method:

Fifth edition: Hassan M.E. Azzazy

spectrometry (HPLC-MS/MS) [2], and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3].

Radioimmunoassay (RIA) has been the most common assay for aldosterone measurement since the early 1970s [4]. The antibodies used were typically polyclonal, raised in rabbits and generated against an aldosterone-3-(O-carboxymethyloxime) bovine–serum albumin conjugate [3]. Commercial immunoassays use different approaches to separate bound from free aldosterone: a solid-phase first antibody, a solid-phase second antibody, an accelerated liquid-phase second antibody, dextran-coated charcoal, or a polyethylene glycol precipitant. RIA methods with and without extraction steps are being employed [5,6]. The extraction of aldosterone prior to analysis may be done into dichloromethane, which improves the specificity of the assay, since aldosterone concentrations in blood are in the picomolar range. Many patients being investigated have some degree of renal failure, and patients with chronic renal failure have polar aldosterone metabolites present in high concentrations in plasma; these cross-react with aldosterone antibodies. Thus solvent extraction helps to increase assay specificity by removing these metabolites. Currently a large portion of RIAs for aldosterone measurement do not involve an extraction, chromatography, or prefractionation step, which are relatively complex and time consuming (Table 1, Methods 1 and 2) [7]. Average salivary aldosterone values are almost a third of those in plasma, and an RIA was also developed for aldosterone determination in saliva [8,9].

Aldosterone measurement in urine is performed using the same testing methodologies, but a preassay acid-hydrolysis step is required. This is because the term urinary aldosterone actually refers to the 18-glucuronide conjugate of aldosterone (10% of all urinary aldosterone metabolites), and acid hydrolysis is required to form free aldosterone. Only 2% of aldosterone is excreted in the free form [4].

Enzyme immunoassays are available for aldosterone, including competitive-binding assays to monoclonal antibodies and direct-detection assays using antibodies conjugated to horseradish peroxidase, with the detection limits being in the picomolar range [9,10]. A competitive-binding chemiluminescence immunoassay has been developed. The assay utilizes a biotinylated mouse monoclonal anti-aldosterone antibody as the capture reagent and acridinium ester-labeled aldosterone as a tracer (Table 1, Method 3) [11].

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is currently being used for aldosterone determination in both blood and urine. Current LC-MS/MS methods use atmospheric-pressure chemical ionization or photospray ionization along with multiple steroid profiling [12,13]. A high-performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC-APCI-MS/MS) method was proposed as a reference method for aldosterone quantification in serum and plasma (Table 1, Method 4). In this method, extraction was performed using dichloromethane/diethyl ether containing flumethasone as internal standard. A phenyl column was used for chromatography, and the mobile phase was 50 mM ammonium formate (pH 7.1)/methanol (50/50, v/v). The reported accuracy of this method ranged from 93.1% to 98.9% [2]. In other LC-MS/MS methods, deuterated aldosterone (aldosterone-d6) is added to samples as an internal control. Aldosterone-d6 and endogenous aldosterone are extracted from serum or plasma using a Strata X cartridge, and the eluate is dried