临床化学分析方法：Aldosterone

Kaplan: Clinical Chemistry, 5th EditionClinical References - Methods of AnalysisAldosteroneHassan M.E. Azzazy iName:AldosteroneClinical significance:click hereMolecular mass: 360.4 DChemical class: MineralocorticoidRefer 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 tablePrinciples of Analysis and Current UsageAldosterone 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 massi AldosteroneNew method:Fifth edition: Hassan M.E. Azzazyspectrometry (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 driedand then reconstituted with 70/30 methanol/water containing estriol before analysis on LC-MS/MS [13]. Recently, an LC-MS/MS method for aldosterone measurement with electrospray ionization has been developed, with a lower limit of detection of 30 pmol/L and interassay coefficients of variation (CVs) between 4.3% and 7.5% at aldosterone concentrations of 97 to 993 pmol/L (Table 1, Method 5) [12].Reference and Preferred MethodsID-GC/MS is the reference method for determination of aldosterone concentration. Although this technique provides reliable results, methods utilizing GC/MS require a labor-intensive and time-consuming sample derivatization step. Liquid chromatography tandem MS (LC-MS/MS) has recently been proposed to replace GC/MS to circumvent the laborious derivatization [12]. Immunoassays are, however, favored for measuring aldosterone in large numbers of small-volume samples. Immunoassays may be problematic unless extracted because of low aldosterone concentration and cross-reactivity.SpecimenSerum, plasma, and urine specimens can be used. For serum, venous blood is to be collected in a glass tube with no additives; plasma can be collected in a glass or plastic tube with the anticoagulants heparin or EDTA. Serum or plasma should be stored frozen and are stable for up to years at −20°C in an airtight container.For urine analysis, 24-hour urine collection is required, and the specimen should be refrigerated during collection. Acetic acid (50% preferred) is added after completion of collection to achieve a pH of 2.0 to 4.0. Other allowed preservatives include thymol and boric acid. Strong mineral acids should not be used. Samples may be stored frozen.InterferencesCross-reactivity with other steroids such as 5-dihydroaldosterone and corticosterone depends on antibody specificity, which varies in different immunoassays. Grossly hemolyzed or lipemic samples or contamination of the sample or tube with 125I or other radioisotopes would interfere with RIA. Also, pharmacological interference was observed in women taking drospirenone, a synthetic progestin with anti-mineralocorticoid activity and aldosterone-receptor antagonism. This drug was reported to interfere with laboratory screening and confirmatory testing for diagnosing primary aldosteronism [15]. Angiotensin, estrogens, laxatives, oral contraceptives, sodium restriction, and thiazide diuretics lead to elevated aldosterone concentration. Aminoglutethimide, ACE inhibitors such as captopril, lisinopril, deoxycorticosterone, prolonged heparin therapy, and saline decrease aldosterone concentration.Reference IntervalsNormal value ranges vary among different laboratories using different assays. It is recommended that each laboratory establish reference ranges for their particular assay. Results of urinary aldosterone depend on the amount of sodium in the diet, where levels are lower with greater intake of sodium. For adults with normal, low-sodium, and high-sodium diets, reported urinary aldosterone levels are 2 to 21, 17 to 44, and 0 to 14 μg/day, respectively. If the 24-hr urinary sodium excretion is > 200 mEq/day, urinary aldosterone should be < 10 μg [16,17].Plasma/Serum Aldosterone Reference Intervals (RIA)Age Position Reference Interval (ng/dL) SI Units(pmol/L)1-12 months — 5-90 139-24931-2 years — 7-54 194-14962-10 years Supine 3-35 83-9702-10 years *Upright 5-80 139-221610-15 years Supine 2-22 55-60910-15 years Upright 4-48 111-1330>15 years Supine 3-16 83-443>15 years Upright 7-30 194-831Adrenal vein — 200-800 5540-22,160Data from Endocrine Sciences. Tarzana, CA: Pediatric Laboratory Services; 1992.* Patient should be seated or standing for at least 2 hours prior to collection of upright specimen.InterpretationElevated concentrations of aldosterone are observed in primary aldosteronism due toaldosterone-secreting adenomas (Conn’s syndrome). Primary aldosteronism is characterized by suppressed renin activity and cannot be stimulated by either sodium restriction or treatment with a diuretic, or by demonstrating lack of suppression of aldosterone following saline infusion or administration of a mineralocorticoid. Pseudoprimary aldosteronism is due to bilateral adrenal hyperplasia. Sampling of adrenal venous renin and aldosterone can be used to differentiate between adenomas and hyperplasia as the cause of hyperaldosteronism. In hyperplasia, bothadrenals secrete high levels of aldosterone. Secondary aldosteronism may be observed in laxative abuse, cardiac failure, diuretic abuse, and Bartter’s syndrome (a rare, inherited defect in the thick ascending limb of the loop of Henle).Aldosterone concentration is important in the assessment of persons with primary hypertension. Primary hyperaldosteronism has recently become accepted as a more frequent cause of hypertension [18]. On the other hand, low aldosterone concentration with hypertension is associated with a number of conditions such as Turner’s syndrome, diabetes mellitus, acute alcoholic intoxication, and excess secretion of deoxycorticosterone. Low concentrations without hypertension are observed in Addison’s disease (primary adrenal insufficiency) and in the syndrome of hypoaldosteronism due to renin deficiency.Measurement of aldosterone concentration by itself is of little value, and irregularities in blood pressure and/or disorders of sodium and potassium homeostasis are best investigated byassessing the integrity of the renin-angiotensin-aldosterone axis [11]. This is usually done by measuring renin and aldosterone concentrations. Although aldosterone measurements alone are difficult to interpret, they may be useful for assessing response of the adrenal cortex tostimulation. The aldosterone-to-renin ratio (ARR) has been proposed as a screening tool for primary aldosteronism. The clinical utility of the ARR remains questionable because of many factors that can affect ARR results. These include posture, time of blood collection, and use of antihypertensive medication. A number of ARR cutoff values have been published, owing to differences in populations studied, collection methods, and hormone assays used. There is no agreed-upon ARR cutoff value, and it is unknown which population should be screened [19,20]. The urinary aldosterone-to-active-renin ratio has been suggested as the best independentpredictor of cure of hypertension after adrenalectomy in patients with aldosterone-producing adenomas [21].Performance GoalsClinical Laboratory Improvement Amendments acceptable performance criteria (CLIA ’88) for measurement of aldosterone require that laboratories be accurate to within ±3 SD of the peer-group mean. Survey data from the College of American Pathologists 2007 participant summary report show imprecision values (% CV) for extracted methods of 14.9% at a mean concentration of 50.2 μg aldosterone/L and 20.3% at a mean concentration of 14.8 μg/L [22].Within-subject and between subject biological variation were 29.4% and 40.1% (serum) and 32.6% and 39% (urine), respectively. Desirable specifications for analytical imprecision derived from studies of biological variation indicate an assay imprecision of no greater than 14.7% (serum) and 16.3% (urine) and a bias of 12.4% (serum) and 12.7% (urine) [23]. References1 Stöckl D, Reinauer H, Thienpont LM, De Leenheer AP. Determination of aldosterone inhuman serum by isotope dilution gas chromatography/mass spectrometry using a newheptafluorobutyryl derivative. Biol Mass Spectrom 1991;20:657-64.2 Fredline VF, Taylor PJ, Dodds HM, Johnson AG. A reference method for the analysis ofaldosterone in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry. Anal Biochem 1997;252:308-313.3 Cawood ML. Measurement of aldosterone in blood. Methods Mol Biol 2006;324:177-85.4 Jaffe BM, Behrman HR, eds. Methods of Hormone Radioimmunoassay. New York:Academic Press; 1979.5 Bayard F, Beitins IZ, Kowarski A, Migeon CJ. Measurement of aldosterone secretion rateby radio-immunoassay. J Clin Endocrinol Metab 1970;31:507-11.6 Jowett TP, Slater JD, Piyasena RD, Ekins RP. Radioimmunoassay of aldosterone inplasma and urine: validation of a novel separation technique and a rapid urine assay. Clin Sci Mol Med 1973;45:607-23.7 Stowasser M, Gordon RD. Aldosterone assays: an urgent need for improvement. ClinChem 2006;52:1640-2.8 Atherden SM, Corrie JE, Jones DB, Al-Dujaili EA, Edwards CR. Development andapplication of a direct radioimmunoassay for aldosterone in saliva. Steroids 1985;46:845-55.9 Hubl W, Taubert H, Freymann E, Hofmann F, Meissner D, Garten CD et al. A simplesolid-phase enzyme immunoassay for aldosterone in plasma and saliva. Exp ClinEndocrinol 1983;82:188-93.10 Hanquez C, Rajkowski KM, Desfosses B, Cittanova N. A competitive microtitre plateenzyme immunoassay for plasma aldosterone using a monoclonal antibody. J SteroidBiochem 1988;31:939-45.11 Schirpenbach C, Seiler L, Maser-Gluth C, Beuschlein F, Reincke M, Bidlingmaier M.Automated chemiluminescence-immunoassay for aldosterone during dynamic testing:comparison to radioimmunoassays with and without extraction steps. Clin Chem2006;52:1749-55.12 Turpeinen U, Hämäläinen E, Stenman UH. Determination of aldosterone in serum byliquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt TechnolBiomed Life Sci 2008;862:113-8.13 Mayo Medical Laboratories Test Catalog. Available at</test-catalog/>14 Perschel FH, Schemer R, Seiler L, Reincke M, Deinum J, Maser-Gluth C et al. Rapidscreening test for primary hyperaldosteronism: ratio of plasma aldosterone to reninconcentration determined by fully automated chemiluminescence immunoassays. ClinChem 2004;50:1650-5.15 Pizzolo F, Pavan C, Corrocher R, Olivieri O. Laboratory diagnosis of primaryaldosteronism and drospirenone-ethinylestradiol therapy. Am J Hypertens 2007;20:1334-7.16 Bravo EL. Primary aldosteronism. Issues in diagnosis and management. Endocrinol MetabClin North Am 1994;23:271-83.17 Young WF Jr. Pheochromocytoma and primary aldosteronism: diagnostic approaches.Endocrinol Metab Clin North Am 1997;26:801-27.18 Mulatero P, Dluhy RG, Giacchetti G, Boscaro M, Veglio F, Stewart PM. Diagnosis ofprimary aldosteronism: from screening to subtype differentiation. Trends EndocrinolMetab 2005;16:114-9.19 Giacchetti G, Ronconi V, Lucarelli G, Boscaro M, Mantero F. Analysis of screening andconfirmatory tests in the diagnosis of primary aldosteronism: need for a standardizedprotocol. J Hypertens 2006;24:737-45.20 Jansen PM, Boomsma F, van den Meiracker AH. Aldosterone-to-renin ratio as a screeningtest for primary aldosteronism: the Dutch ARRAT Study. Neth J Med 2008;66:220-8.21 Mourad JJ, Girerd X, Milliez P, Lopez-Sublet M, Lejeune S, Safar ME. Urinaryaldosterone-to-active-renin ratio: a useful tool for predicting resolution of hypertensionafter adrenalectomy in patients with aldosterone-producing adenomas. Am J Hypertens2008;2:742-7.22 College of American Pathologists. 2007 Survey Participant Summary Report. Northfield,IL: CAP; 2007.23 Ricós C, Alvarez V, Cava F, García-Lario JV, Hernández A, Jiménez CV et al. Currentdatabases on biologic variation: pros, cons and progress. Scand J Clin Lab Invest1999;59:491-500.Clinical References - Methods of Analysis 7-7 Table 1: Characteristics of Selected Aldosterone Assays*Method 1: DSL Active Aldosterone (non-extraction)Sample (volume): Serum/plasma (100 μL)Detection: Polyclonal antibody, 125I tracerRange (ng/L): 2-1600Intraassay variability (%): 3.6-8.3Interassay variability (%): 7.3-10.4Reference interval (ng/L): 30-340 (serum); 30-220 (plasma)*Method 2: Adaltis Aldosterone Maia (non-extraction)Sample (volume): Serum/plasma (50 μL)Detection: Polyclonal antibody, 125I tracerRange (ng/L): 6-2500Intraassay variability (%): 3.5-5.4Interassay variability (%): 3.6-6.4Reference interval (ng/L): 70-350 (plasma)*Method 3: Nichols Advantage Aldosterone [14]Sample (volume): Serum/plasma (450 μL)Detection: Monoclonal antibody, chemiluminescenceRange (ng/L): 15-1200Intraassay variability (%): 2.9-14.0Interassay variability (%): 4.9-18.6Reference interval (ng/L): 38-313 (serum)Method 4: High-performance liquid chromatography atmospheric pressurechemical ionization–tandem mass spectrometry (HPLC-APCI-MS/MS) [2]Sample: Serum/plasmaLimits of detection and quantification (pg/mL): 10 and 15Linear range (pg/mL): 15-500Assay imprecisions at 15, 20, 150, and 450 pg/mL were 18.5%, 8.8%, 10.6%, and9.5%Method 5: Liquid chromatography–tandem mass spectrometry (LC-MS/MS)[12]Sample (volume): Serum (500 μL)Lower limit of detection (pmol/L): 30Linear range (pmol/L): 60-3000Interassay variability (%): 4.3-7.5。