Antiadrenal Antibody

Under normal circumstances, 21-hydroxylase antibodies should not be detected in serum samples. A positive test result is generally consistent with the presence of primary adrenal insufficiency (Addison disease).[1, 2, 3]

Positive results for the 21-hydroxylase antibody assay indicate the presence of adrenal cortex autoantibodies in serum, a finding that is consistent with Addison disease.[1]

Specifics for the handling of specimens are as follows:

• Preferable specimen - Serum (fresh or frozen)

• Container - Red-top tube or serum separator tube (SST)

• Minimum sample volume - 1 mL

• Temperature for storage and transport - Fresh serum is acceptable if testing is performed immediately; if testing is delayed, specimen should be frozen.

• Hemolyzed and lipemic specimens are unacceptable

• Specimens should be centrifuged and cells should be separated as soon as possible after collection

• Stability (from collection to initiation of testing, if testing is delayed) - 1 week (refrigerated); 6 months (frozen)

Measurement of 21-hydroxylase–specific antibody

The 21-hydroxylase antibody assay is the main means of evaluating adrenal autoimmunity. Up to 90% of persons with Addison disease have autoantibodies against 21-hydroxylase. Therefore, the detection of these antibodies can clarify the root cause of adrenal insufficiency.

The test, which is qualitative, employs an enzyme-linked immunosorbent assay (ELISA). 

Patient specimens and corresponding controls, as well as a reference preparation, are incubated overnight on a plate coated with 21-hydroxylase biotin, to which the sample's 21-hydroxylase antibodies bind. The addition of streptavidin peroxidase (SA-POD) and tetramethylbenzidine (TMB) allows detection of the resulting antigen-antibody-antigen complexes, with a colorimetric complex produced. The absorbance of each well is in direct proportion to the existing amount of antibody.[4]

In the United States, the assay can be obtained through Kronus, Inc, and is available in many reference laboratories.

Description

Steroid synthesis is a very complex process that takes place in the adrenal glands and gonads. The pathway involves multiple enzymes and multiple enzymatic reactions. Steroid synthesis starts from cholesterol (low-density–lipoprotein cholesterol [LDL-C]) and leads to production of three classes of specific adrenocortical steroid hormones: glucocorticoids, mineralocorticoids, and sex steroids. See the figure below.

Steroid hormones pathway.

Deletion or mutation of the genes encoding the enzymes involved in the synthesis or impaired function of the enzymes involved in the steroid hormone pathway (eg, via autoantibodies) leads to a deficiency of different hormones.[5]

Adrenal insufficiency causing combined mineralocorticoid and glucocorticoid deficiency is rare, with a prevalence of only 4 to 11 cases per 100,000.[6]  In developing countries, infections (eg, tuberculosis, syphilis, human immunodeficiency virus, fungal infections) are historically the most frequent cause of adrenal insufficiency. In industrialized nations, the main cause of adrenal insufficiency is autoimmunity, particularly in relation to autoantibodies against 21-hydroxlyase. Specifically, autoimmune adrenalitis accounts for more than 70% of cases of primary adrenal insufficiency (Addison disease) reported in the Western world, with adrenal autoantibodies measurable in more than 75% of cases. Addison disease often exists as one of the morbidities of autoimmune polyglandular syndrome (APS), either APS I or II.[7]

APS I is characterized by Addison disease, candidiasis, hypoparathyroidism, and primary gonadal failure, while APS II is characterized by Addison disease, primary hypothyroidism, primary hypogonadism, diabetes, and pernicious anemia.

Antibodies against the 21-hydroxylase enzyme or other intracellular antigens have been used to diagnose autoimmune adrenal insufficiency. When, in immunofluorescence tests, all layers of the adrenal cortex fluoresce, this indicates that an elevated level of adrenal cytoplasmic autoantibodies (ACAs) is in the circulation.[8] It is estimated that about 60-70% of patients with Addison disease express ACAs. Adrenal antibodies targeting steroidogenic enzymes—including P450scc (CYP11A1, side-chain cleavage enzyme), P450c17 (CYP17, 17-alpha-hydroxylase), and P450c21 (CYP21A2, 21-hydroxylase)—are responsible for humoral adrenal autoimmunity. Patients with autoimmune adrenal disease predominantly have autoantibodies directed against the microsomal 21-hydroxylase. Therefore, laboratory assessment of such antibodies is clinically useful for the diagnosis of autoimmune adrenal disease (eg, Addison disease).[1, 9, 10, 11]  They can be evaluated in serum via immunoprecipitation or ELISA.

A study by Wolff et al indicated that individuals with autoimmune Addison disease have long-term positivity for 21-hydroxylase–specific antibodies, with the investigators finding that more than 90% of the study’s patients were still positive for the antibodies 30 years after their disease diagnosis. According to the report, indices for the antibodies are impacted by disease duration, sex, whether the Addison disease is isolated or part of APS type I or II, and human leukocyte antigen–risk genotype.[12]

Indications/applications

Antibodies to 21-hydroxylase are markers of autoimmune Addison disease, whether this condition is occurring alone or in conjunction with other autoimmune endocrine diseases as part of type I or type II autoimmune polyglandular syndrome (APS). These antibodies may be present even before any reduction in endocrine function is apparent. Patients with autoimmune adrenal insufficiency commonly have antibodies against other endocrine glands, whereas healthy patients rarely do. More than 50% of these patients have high serum antithyroid peroxidase antibody levels, and nearly 50% have overt hypothyroidism. Many more of these patients exhibit subclinical hypothyroidism (evidenced by elevated thyroid-stimulating hormone [TSH] and normal thyroxine concentrations) and are at risk for subsequent development of overt hypothyroidism (Schmidt syndrome).[13, 14]

Autoimmune adrenal disease can be divided into phases. Initially, the adrenals may be enlarged, and extensive lymphocytic infiltration may be noted. In long-standing disease, however, the glands are small and may be hard to find. A thickened, fibrotic capsule is observed, with complete destruction of the cortex (although a few small clusters of adrenocortical cells surrounded by lymphocytes may remain) and relative sparing of the medulla. Only after at least 90% of the cortex has been destroyed does adrenal insufficiency clinically manifest.

Considerations

In patients with normal adrenal function who have other autoimmune endocrine diseases, the prevalence of antiadrenal autoantibodies in serum is low (2%), the exception being those with hypoparathyroidism, in whom the figure is substantially higher (16%).[15, 16]

Autoimmune adrenal insufficiency may be either familial or nonfamilial (acquired); when occurring alone, rather than as part of APS type I or II, it is somewhat less likely to be familial. About half the patients who have adrenal insufficiency as part of APS type I or II have affected family members, compared with only about one third of patients who have autoimmune adrenal insufficiency alone.

As the enzyme 21-hydroxylase is involved in the metabolism of both mineralocorticoids (aldosterone) and glucocorticoids (cortisol) (see figure above), impaired 21-hydroxylase function due to the binding antibodies can lead to deficiency in both of these types of hormones. On the other hand, patients have an elevated serum concentration of 17-α hydroxyprogesterone, progesterone, 17-α hydroxypregnenolone, and pregnenolone. Adrenal androgens, dehydroepiandrosterone sulfate (DHEA-S), DHEA, and 3-α androstanediol (a metabolite of androgens) are also often elevated in these patients.

Women might present with hirsutism (mild), polycystic ovarian syndrome (PCOS) and primary and secondary amenorrhea. The adrenals might become hyperplasic and the patient might have low serum sodium and chloride levels and hyperkalemia (salt-wasting forms).[5]