Diagnostic Testing in Cushing's Syndrome: Reassessment of 17-hydroxycorticosteroid and 17-ketosteroid Measurements
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1 CRTCAL REVEW [ K e i t h D u n c a n, M. D. March, 1985 Diagnostic Testing in Cushing's Syndrome: Reassessment of 17-hydroxycorticosteroid and 17-ketosteroid Measurements ntroduction The measurement of 17-ketosteroids (17-KS) and 17-hydroxy corticosteroids (17-OHCS) in timed, 24-hour urine collections has been important in the evaluation of adrenal function. 17-KS have been utilized as a general index of androgen secretion while 17-OHCS have been used as a gauge for Cortisol secretion. Recently, assays of individual steroids have gradually replaced these tests due to their increased specificity and sensitivity. 17-KS are those steroid compounds with a keto-group at the C-17 position. n females, this steroid group is produced essentially by the adrenal glands. n males, approximately two-thirds of 17-KS are derived from the adrenals while the remainder is from the testis. ncreased urinary excretion of 17-KS may be observed in Cushing's syndrome, in certain adrenal, testicular and ovarian tumors, and congenital adrenal hyperplasia syndromes. The methodology includes removal of sulfate or glucuronide conjugating groups followed by reaction of the steroid with m-nitrobenzene (Zimmerman reaction). A red-purple color results that is measured spectrophotometrically at 520 nm. The compounds measured in the 17-KS determination include androstenedione, etiocholanolone, dehydroepiandrosterone, and their metabolites. 17-OHCS are those steroid compounds which contain 21 carbon atoms and have a dihydroxyacetone side chain. This determination has been useful for estimating glucocorticosteroid excretion in the diagnosis of Cushing's syndrome and Addison's disease. The methodology is based on the reaction of these compounds with phenylhydrazine to produce a color that absorbs at 410 nm (Porter-Silber chromogens). The compounds measured by this technique include 11-deoxycortisol, Cortisol, cortisone, and cortisone metabolites. Technical Problems The measurements of 17-KS and 17-OHCS are complicated by many factors, as outlined below (Rudd, 1983): 1. Precision is poor since the coefficients of variation between different labs are quite high compared to radioimmunoassay (RA) for individual steroid hormones. 2. Specificity is poor due to other chromogens; numerous drugs (aspirin, ascorbic acid, tranquilizers, diuretics, antibiotics, etc.) interfere with the assay or with metabolism to give spurious results (Bonishek and Gold, 1964). Specificity is also poor due to numerous false positives in obese patients using 17-KS and 17-OHCS measurements.
2 c Duncan/2 Eddy et al. (1973) reported that "Cushingoid obesity" patients had levels of both 17-OHCS and 17-KS that were falsely elevated in 53% (8/15) and 62% (9/15), respectively. Also, patients with Cushing's syndrome had 17% (4/24) false negatives with either steroid measurement. n contrast, there were no false negatives in 24 Cushing's syndrome patients and no false positives in 15 "Cushingoid obesity" patients utilizing urinary free Cortisol. 3. Measurements in neonatal specimens are unreliable due to the low concentration of these compounds and poor sensitivity of the methodology. 4. Total costs for the laboratory are high due to labor-intensive procedures. 5. Solvents and reagents are potentially hazardous (ether, m-nitrobenzene). Clinical Applications The diagnosis of Cushing!s syndrome relies on the demonstration of sustained Cortisol overproduction by: 1. Dexamethasone suppression tests with non-suppressible serum Cortisol levels. 2. ncreased urinary free Cortisol (UFC) excretion. ff^- Overnight, low dose, and high dose dexamethasone suppression tests of serum Cortisol levels are utilized in the workup of CushingTs syndrome. The majority of authors reviewed first do an overnight dexamethasone suppression test of an a.m. serum Cortisol level, followed by a UFC to confirm a positive test (see Appendix). Crappo (1979) computed 1.9% false negative results in patients with Cushing!s syndrome (3/154) using the overnight dexamethasone suppression test by combining data of 15 separate studies. False positives with this test were only 1.1% in normal outpatient controls but increased to 13% and 23% in obese and chronically ill patients, respectively. Thus the overnight dexamethasone suppression test is an excellent screening device for suspected Cushing's syndrome which may require confirmation with UFC levels. UFC levels are an important index of adrenocortical hyperfunction for a 24-hour integrating measurement of steroid metabolites which undergo diurnal variation and pulsatile release. Crappo (1979) stated that UFC levels are a superior test compared with 17-OHCS or 17-KS, particularly in obese patients. Crappo combined the data of 14 separate studies to show that there were 11% false negatives (315 patients with CushingTs syndrome) and 27% false positives (173 control patients with obesity) utilizing 17-OHCS mesurements. n contrast, 5.6% false negatives (248 patients with CushingTs syndrome) and 4.9% false positives (104 patients with obesity) were found using UFC excretion in 15 separate studies. UFC can be determined by either RA or high pressure liquid chromatography (HPLC). Due to interference by cross-reacting substances in urine, the RA method tends to overestimate Cortisol concentrations by a three- to four-fold factor compared to HPLC results. Normal reference ranges vary depending on the
3 Duncan/3 j ^ N particular methodology and thus are established by each laboratory (Schoneshofer et al., 1985). Zadik (1980) reported 11% false negatives and 24% false positives utilizing 17-OHCS measurements in 13 patients with Cushing's syndrome and 40 controls with mild hypertension. UFC excretion in these patients revealed 5% false negatives and 3% false positives. Recently, Dunlap et al. (1985) reported no false negatives or false positives with UFC levels in the study of 18 patients with Cushing's syndrome and 25 patients in which Cushing's syndrome was excluded in follow-up. 17-OHCS measurements had 16% (2/12) false negatives but no false positives in this study. The diagnosis of Cushing's syndrome was confirmed histologically in 15 patients while 3 patients had invasive pituitary tumors on CT scan. Summary 17-KS and 17-OHCS measurements have been used in the past to evaluate androgenic states and adrenal cortical function, respectively. However, with the availability of specific steroid metabolite measurements by RA or HPLC, 17-KS and 17-OHCS assays have become obsolete. The inherent technical problems of these urinary steroid measurements were outlined above. Urinary free Cortisol is a more reliable test in evaluating Cortisol excretion than either 17-OHCS or 17-KS measurements and should replace these other assays. 17-KS and 17-OHCS measurements have also been replaced by other steroid assays in the evaluation of adrenal insufficiency, congenital adrenal hyperplasias, and polycystic ovary disease. References 1. Aron DC, et al.: Cushing's Syndrome. Problems in Diagnosis. Medicine 60:25-35, Bonishek S, Gold JJ: Commonly Used Medications that nterfere with Routine Endocrine Laboratory Procedures. Clin Chem 10:41-52, Crappo L: Cushing!s Syndrome: A Review of Diagnostic Tests. Metabolism 28: , Dunlap N, Grizzle WE, Siegel AL: Cushing's Syndrome: Screening Methods inhospitalized Patients. Arch Pathol Lab Med 109: , Eddy RL, et al.: Cushingfs Syndrome: A Prospective Study of Diagnostic Methods. Am J Med 55: , Gold EM: The Cushing Syndrome: Changing Views of Diagnosis and Treatment. Ann ntern Med 90: , Rudd B: Urinary 17-oxogenic and 17-oxysteroids: A Case for Deletion from the Clinical Repertoire. Ann Clin Biochem 20:65-71, 1983.
4 r Duncan/4 8. Schoneshofer M, et al.: Determination of Urinary Free Cortisol by "On-Line" Liquid Chromatography. Clin Chem 31: , Zadik Z, et al.: A Comparative Study of Urinary 17-OHCS, Urinary Free Cortisol, and the ntegrated Concentration of Plasma Cortisol. J Clin Endocrinol Metab 51: , 1980.
5 $^*\ APPENDX SUSPECTED lmg OVERNGHT DEXAMETHASONE SUPPRESSON TEST ABNORMAL am plasma Cortisol >5 ug/dl CONFRM WTH 24 hr URNARY FREE CORTSOL(UFC) NORMAL am plasma Cortisol <5 ug/dl 1 EXCLUDED (unless clinical evidence is overwhelming) V UFO 200 ug/24 hr UFC ug/24 hr UFC <100 ug/24 hr DAGNOSED EXCLUDED HGH DOSE DEXAMETHASONE SUPPRESSON TEST UFC OR PLASMA CORTSOL NON-SUPPRESSBLE<50% BASELNE DAGNOSED UFC OR PLASMA CORTSOL SUPPRESSBLE i EXCLUDED FURTHER WORK-UP WTH CAT SCANS, PLASMA ACTH, ETC.
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