Clarke Halfman, Ph.D.

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Review of Normal Physiology
Altered Function in Adrenal Disease
Laboratory Diagnosis of Adrenal Disease
Conn's disease
    - Tests for Evaluating Dysfunctional Cortisol Metabolism
Congenital Adrenal Hyperplasia

I. Review of Normal Physiology
The two anatomically distinct regions of the adrenal glands elaborate hormones with different chemical structures and different physiolologic effects. The medulla elaborates the catecholamines, adrenaline and noradrenaline, one significant effect of which is to ele vate blood pressure. The cortex elaborates steroids which are referred to on the basis of their major physiologic effects, glucocorticoids and mineralcorticoids.
Normally, the major glucocorticoid is cortisol and the major mineralcorticoid is aldosterone. The principal pathway for adrenal steroid synthesis is shown below. (Click on the figure to view a more detailed pathway.)

ACTH stimulates adrenal steroid synthesis. Circulating concentrations of cortisol are maintained within the normal range by the negative feedback effect of cortisol on pititary ACTH elaboration. Aldosterone synthesis is not stimulated by ACTH but by angiotensin II, although ACTH does stimulate synthesis of aldosterone precursors. Circulating Potassium exerts a permissive effect on angiotensin II stimulation; high potassium enhances and low potassium diminishes.

In pathologic conditions, adrenal synthesis of precursor steroids may be greatly stimulated by high ACTH concentrations so that circulating concentrations of cortisol precursors may be sufficiently high to exhibit glucocorticoid activity and/or the circulating concentration of 11-deoxycorticosterone may be sufficiently high to exhibit mineralcorticoid activity.
17-keto steroids are by-products of adrenal steroid synthesis and are also synthesized at increased rates by ACTH stimulation. High circulating concentrations of dehydroepiandrosterone exhibit androgenic activity.

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II. Altered Function in Adrenal Disease


The symptoms of adrenal disease result from abnormal circulating hormone concentrations because of altered function. The abnormal functional state associated with specific disease entities is summarized in the table to the right.

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III. Laboratory Diagnosis of Adrenal Disease

A. Pheochromocytoma/Neuroblastoma (increased catecholamines)
Determination of increased circulating concentration of catecholamines is technically difficult and is not commonly performed. Metanephrines and VMA are degradation products of the catecholamines and their determination in urine specimens is more commonly done.

catecholamines ---> metanephrines ---> VMA

Determination of VMA is simplest but suffers from false positives. Analysis of metanephrines is more specific and is done to confirm increased VMA.

B. Conn's disease (Increased aldosterone & decreased renin)
Serum concentrations of aldosterone and renin are not routine tests but are determined in reference laboratories by radioimmunoassay. In primary hyperaldosteronism, serum concentrations of renin are decreased in contrast to other causes of hypertension not involving adrenal dysfunction. Renin is also decreased in Cushing's syndrome and in some cases of congenital adrenal hyperplasia because of hypertension from increased plasma concentrations of the aldosterone precursor, 11-deoxycorticosterone, having only weak mineral corticoid activity at normal concentrations.

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C. Dysfunctional Cortisol Metabolism

The following tests are used to evaluate abnormal cortisol metabolism.

             Test                         Normal Range          
        serum cortisol                 
           8 AM                         7-25 ug/dl
           4 PM                          ~ one-half the AM value

        urine free cortisol            30-100 �g/24 hrs.

        17 hydroxy corticosteroids      3-12 mg/24 hrs.

        17 ketosteroids
            male                       10-25 mg/24 hrs.
            female                      5-15

        17 ketogenic steroids
            male                        8-25 mg/24 hrs.
            female                      5-18

       *plasma ACTH - 8 AM             20-100 pg/ml

*Technical difficulties had precluded routine measurement of ACTH by radioimmunoassay until recently. To obtain information on pituitary status, dexamethasone, or metyrapone or ACTH was administered and the influence on plasma cortisol, or urinary steroids, was determined. These indirect inhibition/stimulation tests for assessing pituitary status in regard to ACTH elaboration are still in use.

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Total serum cortisol is determined by radioimmunoassay. Interpretation is complicated by a normal diurnal variation, and by elevation of total concentrations because of variations in the circulating concentration of the transport protein (cortisol binding globulin - CBG) for the same reasons that TBG is variable. These interpretive complications are overcome by collecting specimens for cortisol determination in the morning and the late afternoon or early evening, thereby taking advantage of the normal diurnal variation. Results are best interpreted in relative, rather than absolute terms, i.e. results are considered normal if the result from the late afternoon specimen is about 1/2 the value of the morning specimen, even if the absolute values are elevated or depressed. However, intermittent, transient stress induced elevations still confound unambiguous interpretation of results. Measurement of free cortisol in a 24 hour urine specimen abrogates these difficulties.
Urine free cortisol determination involves specific measurement by radioimmunoassay of unmetabolized, unconjugated cortisol in an aliquot of a 24 hour urine specimen. Test results overcome the interpretive difficulties associated with serum cortisol results because only free serum cortisol is filtered by the glomeruli and excreted in the urine, so that abnormal CBG concentrations do not affect results, and the relatively long collection period minimizes the effect of intermittent, transient stress induced elevations.
Determination of free cortisol in a 24 hour urine specimen is considered the best test for evaluating adrenal function in regard to corticosteroid elaboration.

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The other three urine steroid assays measure different groups of parent steroids and their metabolic waste products as illustrated in the figure below. Parent steroids and metabolic waste products of the mineralcorticoids are quantitatively insignificant, compared to the 17-ketosteroids and the steroids in the glucocorticoid pathway, and do not contribute to assay results.

The 17-Hydroxy Steroids assay measures reduced products of cortisol and its immediate precursor, 11-deoxycortisol, excreted in urine. The results provide an indication of the activity of the glucocorticoid pathway (amount of steroids catabolized and excreted = synthetic activity). Normally, the catabolic products of cortisol are the major steroids measured. In cases of 11-hyroxylase deficiency or when metyrapone is administered diagnostically to inhibit 11-hydroxylase activity, then 11-deoxycortisol and its catabolic products are substantially increased and contribute to the measurement.

The 17-ketosteroid assay measures the 17-ketosteroids. The major urine 17-ketosteroids are dehydroepiandrosterone and the reduced catabolic product of Δ-4-androstenedione, androsterone. The assay results indicate the activity of the androgen pathway.

The 17-ketogenic assay measures all steroids, and reduced catabolic products, with oxygen atoms on carbons 17 and 20; i.e., all of the steroids in the glucocorticoid pathway. (In contrast, the 17-hydroxycorticosteroid assay only measures cortisol and 11-deoxycortisol and reduced catabolic products.) The assay is useful in investigating enzyme deficiencies in congenital adrenal hyperplasia.

Parent Steroids Whose Reduced and Conjugated Products are Measured in Urine Assays

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1. Laboratory Diagnosis of Hypocorticism

Etiology cortisol ACTH
primary adrenal insufficiency - most common
pituitary deficiency - less frequent
iatrogenic - atrophic adrenal and pituitary hypofunction from long term, high dose steroid therapy.

Definite cases exhibit low serum cortisol concentrations with loss of diurnal variation. If serum results are equivocal, 24 hour urine free cortisol would be conducted and found to be low.
Because of the great functional reserve of the adrenal cortex, greater than 90% of tissue must be destroyed before clinically obvious symptoms develop and plasma and urine steroid levels are subnormal. Suspected borderline cases are confirmed by testing the reserve capacity of the adrenals by metyrapone inhibition, or hypoglycemic "stress" induced by insulin administration, or by directly stimulating with ACTH.
  1. Metyrapone inhibition - The drug (4.5 g) is administered in divided doses over a 24 hour period while a urine specimen is collected for 17-OH corticosteroid determination. Results are compared to a control 24 hour urine specimen collected the day before the test. Metyrapone inhibits 11-hydroxylase activity so that plasma cortisol decreases and 11-deoxycortisol accumulates. The normal response is increased elaboration of ACTH by the pituitary and maximal stimulation of steroid synthesis so that urine steroids are increased 2-5 fold. Less than 2 fold stimulation is consistent with either primary or secondary insufficiency.
  2. Hypoglycemic stimulation - induced by insulin administration
    Plasma glucose is decreased below 40 mg/dl by insulin administration and blood specimens are collected at 30, 60 and 90 minutes for determination of cortisol. Hypoglycemia induces elaboration of ACTH by the pituitary, so that the normal response is an increase in plasma cortisol of at least 7 ug/dl in each timed specimen or a rise to at least 20 ug/dl in the 90 minute specimen. A smaller increase is consistent with either primary or secondary insufficiency.
  3. ACTH stimulation:
    • rapid test - A blood specimen for cortisol determination is collected before and 30-60 minutes after injection of 25 units of ACTH. The normal response is an increase in plasma cortisol by at least 7 ug/dl. A diminished or absent response is found in both primary as well as secondary adrenal insufficiency (because of disuse atrophy).
    • prolonged test - Hourly blood specimens are collected for plasma cortisol for 5 hours after I.M. injection of slow release (depot) ACTH. Normal response is a continuous increase in plasma cortisol levels up to at least 35 ug/dl in the five hour specimen. In primary Addison's disease, no response occurs. In secondary adrenal insufficiency, with atrophic hypofunction, the response may be diminished, but not absent, unless the condition is severe and long standing.

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2. Laboratory Diagnosis of Hypercorticism (Cushing's syndrome)

Etiology cortisol ACTH
secondary to pituitary hypersecretion of ACTH (Cushing's disease) ~ 70% / N
secondary to ectopic ACTH secretion ~ 15%
primary adrenal tumor ~ 15%

Definite cases exhibit increased serum cortisol concentrations with loss of diurnal variation, increased free cortisol in 24 hour urine specimens, increased 24 hour urine 17-OH corticosteroids, as well as other characteristic laboratory findings such as hyperglycemia, hypertension, hypokalemia and mild alkalosis. Borderline cases and distinction between primary and secondary hyperfunction require further evaluation:
  1. Evaluation of borderline cases (low dose dexamethasone test)
    Single, low dose dexamethasone test: Blood specimens for plasma cortisol determination are collected at 8:00 A.M. and 4:00 - 8:00 P.M. One mg of dexamethosone is administered at 11:00 P.M. and another blood specimen is collected at 8:00 A.M.
    • Normal response:   second A.M. specimen is ~ 50% less than first A.M. specimen.
    • Response in Cushing's syndrome - second A.M. specimen is not reduced.

  2. Differentiation of pituitary hypersecretion of ACTH, ectopic ACTH secretion and primary adrenal tumor:
    Pituitary hypersecretion of ACTH can be differentiated from ectopic ACTH secretion and adrenal tumor most readily on the basis of results from ACTH determination. Before reliable ACTH determinations were available, the differentiation was based on results from the ACTH stimulation test, the Metyrapone test and the high dose Dexamethasone suppression test.
    Results in Hypercorticism

    Interpretation of results is based on the following general behavior:

    • Pituitary Cushing's -
      ACTH is not elaborated autonomously; the pituitary responds to circulating cortisol concentrations but with a higher "set point" for "turning off" ACTH secretion. Also, ACTH is elaborated in greater amounts at low cortisol concentrations.
    • Ectopic ACTH -
      tumors elaborate ACTH autonomously and generally at higher rates than the hyperfunctioning pituitary in Cushing's disease; atrophic hypofunctioning of the normal pituitary occurs.
    • Primary adrenal tumor -
      elaborates cortisol autonomously; the normal pituitary and adrenal suffer disuse atrophy.

    Before the advent of reliable ACTH determination, the high dose dexamethasone supression test was most commonly used to evaluate etiology. Suppression occurs only in cases of pituitary Cushing's. Ectopic tumors are not turned off by dexamethasone; nor, of course, are primary adrenal turmors.
    It should be clear why each condition responds as it does in each of the other tests.

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3. Laboratory Evaluation of Enzyme Deficiencies in Congenital Adrenal Hyperplasia

In cases of congenital adrenal enzyme deficiency, increased, compensatory pituitary ACTH elaboration causes adrenal hyperplasia. In most cases, the enzyme deficiency is partial and the compensatory hyperplasia results in near normal circulating cortisol concentrations. Symptoms (hypertension and/or virilism) are caused by accumulation of steroid precursors proximal to the deficient enzyme. Accumulation of 11-deoxycorticosterone accounts for hypertension and accumulation of dehydroepiandrosterone (DHEA) accounts for virilism.
The symptoms associated with the different enzyme deficiencies and the corresponding findings of urine steroid assays are summarized in the table below.

Approximately 95% of all cases of congenital adrenal hyperplasia are due to partial deficiency of the 21-β-hydroxylase. Compensatory, increased ACTH stimulation allows near normal cortisol synthesis and a sufficient quantity of mineral corticoid precursors for normal aldosterone synthesis. Precursors proximal to the deficiency accumulate and increased concentrations of DHEA cause virilism in females or precocious puberty in males. The urinary 17-ketosteroids and 17-ketogenic steroids are increased and the 17-hydroxycorticosteroids are near normal or only slightly decreased.

Most of the other 5% of cases of congenital adrenal hyperplasia are due to partial deficiency of the 11-β-hydroxylase. The ACTH stimulated compensatory hyperplasia permits near normal synthesis of cortisol and aldosterone, but the increased precursors, 11-deoxycorticosterone and DHEA, cause hypertension and virilism (or precocious puberty), respectively. Results from all three urine steroid assays are increased. Increased 11-deoxycortisol accounts for the increased 17-hydroxycorticosteroids.

Partial deficiency of the 17-hydroxylase occurs only rarely. ACTH stimulated hyperplasia allows near normal synthesis of glucocorticoids and androgens. The mineral corticoid pathway is hyperstimulated and increased concentration of 11-deoxycorticosterone causes hypertension. Results from all three urine steroid assays are near normal or slightly decreased.

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Last Updated: March 28, 2001