Diagnosing hypercortisolism.

Cortisol, a glucocorticoid hormone, plays an essential role in adaptation to stress, regulation of metabolism, and inflammatory responses. Hypercortisolism is not only associated with a rare syndrome known as Cushing's but also has been linked to hypertension, diabetes, obesity, and pseudo-Cushing's.

Analysis of free cortisol in serum/plasma, urine, and saliva is used for the diagnosis of hypercortisolism. Recent studies indicate that increase in salivary cortisol at the midnight cortisol (cortisol of nadir) appear to be one of the earliest abnormalities in Cushing's compared to 24-hour urinary and random plasma cortisol. The clinical outcome in patients with Cushing's is very positive when treated by expert endocrinologists and radiologists, and when experienced surgeons remove the pituitary or adrenal tumor. It is also well established that to distinguish Cushing's from pseudo-Cushing's is very difficult in the setting of mild to moderate hypercortisolism. To emphasize further distinguishing pituitary-dependent Cushing's disease from pseudo-Cushing's states presents a diagnostic challenge. Currently, diagnosis of a pseudo-Cushing state is normally based on negative tests for Cushing's, including a dexamethasone-CRH (corticotropin-releasing hormone) stimulation test, a loperamide suppression test, and/or a 1-mg overnight dexamethasone-suppression test, and/or resolution of hypercortisolism over the course of up to 18 months' follow-up.

Cortisol primarily binds to intracellular receptors leading to altered target gene transcription of proteins responsible for metabolism. Cortisol is also shuttled between active and inactive forms by tissue- specific enzymes. The latter effect is accomplished by two different 11 beta-hydroxysteroid dehydrogenase isoenzymes (11[beta]-HSD), constituting a system between the receptor ligand cortisol and its non-binding precursor cortisone. The type 2 enzyme is an exclusive NA[D.sup.+]-dependent dehydrogenase of glucocorticoids, thus "protecting" the mineralocorticoid receptor against cortisol. 11[beta]-HSD-2 is mostly present in the kidneys, whereas 11[beta]-HSD-1 is present in the liver, muscle, and adipose tissue with activity being higher in omental than in subcutaneous fat. Inhibition or knockout of 11[beta]-HSD-1 in mice improves hepatic insulin action and protects against obesity and hyperglycemia. Conversely, overexpression of 11[beta]-HSD-1 in adipose tissue of mice results in visceral obesity, hyperglycemia, hyperlipidemia, and hypertension. There is a negative correlation between the extent of inactivation of cortisol to cortisone (inhibition of 11[beta]-HSD-2) and elevation of blood pressure. Similarly, selective hepatic overexpression of 11[beta]-HSD-1 causes insulin resistance and hypertension. In contrast to mice, however, the effect of obesity or diabetes on production of cortisol by 11[beta]-HSD-1 in humans has been more controversial. (1) Urinary ratios of cortisol to cortisone metabolites (commonly used to estimate whole-body 11[beta]-HSD-1 activity) have been reported to be increased, decreased, or not different in obese non-diabetic or diabetic subjects compared with lean non-diabetic subjects. The latter discrepancies likely have been observed--at least, in part--because urinary ratios of cortisol and cortisone metabolites are influenced by multiple factors, including the pattern of cortisol metabolism.

Historically, various radioimmunoassay (RIA) methods have been used for analysis of free cortisol in urine and plasma. RIA has been replaced with more sensitive and specific chemiluminescent immunoassay (CIA) methods. Even though all immunoassays for free cortisol use extraction methods to eliminate interfering compounds, these methods are still prone to interferences from other endogenous steroid metabolites and exogenous synthetic glucocorticoids. The extraction procedure used with immunoassays does not correct for variable recovery of cortisol from different specimen types of serum/plasma, urine, and saliva compared to chromatographic methods. The limitations of immunoassays for free cortisol have led to development of more specific liquid chromatography-ultraviolet (LC-UV) and liquid chromatography-mass spectrometry (LC-MS) methods.

[ILLUSTRATION OMITTED]

The LC-UV methods for cortisol and cortisone require lengthy analysis time to obtain resolution between cortisol and cortisone, and to assure that the commonly used synthetic corticosteroids and the more hydrophilic cortisol metabolites do not interfere with the cortisol and cortisone peaks. Despite its advantages over immunoassays, LC-UV methods are still prone to some interferences, the most notable being carbamazepine and its hydroxy metabolites. To resolve the carbamazepine interferences, LC-MS or gas chromatography-mass spectrometry (GC-MS) analysis is required. Although chromatographic methods with MS detection provide specific quantitation of cortisol, they have not been widely implemented due to low throughput and higher cost of instrumentation.

The index of 11[beta]-HSD-2 activity in humans is currently monitored by calculating the ratio of cortisol and cortisone-reduced metabolites in the urine by the GC-MS method. The GC-MS method used for analysis of these metabolites involves hydrolysis, extraction, and derivitization, and has very low throughput. Recently, various publications have indicated that direct cortisol/cortisone ratio is a better indicator of 11[beta]-HSD activity than the ratio of urinary metabolites of cortisol and cortisone. Simultaneous analysis of free cortisol and cortisone by the LC-tandem mass spectrometry (MS/MS) method is helpful for diagnosis of Cushing's and apparent mineralocorticoid excess (AME) syndrome. The syndrome of AME is an inherited form of hypertension. This disorder results from an inability of the enzyme 11[beta]-HSD-2 which inactivates cortisol to cortisone. Cortisol, like aldosterone, has mineralocorticoid properties but circulates in much higher concentration. AME patients with 11[beta]-HSD-2 deficiency may have cortisone to cortisol ratios <1, whereas, a ratio of >2 is observed in normal individuals. Excessive licorice consumption and carbenoxolone (a synthetic derivative of glycyrrhizinic acid used to treat gastroesophageal reflux disease) will also suppress the ratio to <1.

The recent introduction of MS/MS in the clinical laboratory has overcome the limitations of the LC-UV, LC-MS, and GC-MS methods for the analysis of various clinically relevant analytes. Various clinical laboratories are now using LC-MS/MS method, which not only provides simultaneous analysis of free cortisol and cortisone but also has high throughput and least interferences. (2) The implementation of LC-MS/MS methodology is step forward in improving the diagnosis of Cushing's from pseudo-Cushing's.

References

(1.) Basu R, Singh RJ, Basu A, Chittilapilly EG, Johnson CM, Toffolo G. Cobelli C, Rizza RA. Splanchnic cortisol production occurs in humans: evidence for conversion of cortisone to cortisol via the 11-beta hydroxysteroid dehydrogenase (11 beta-hsd) type 1 pathway. Diabetes. 2004;53:2051-2059.

(2.) Taylor RL, Machacek D, Singh RJ. Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem. 2002;48:1511-1519.

By Ravinder J. Singh, PhD

Ravinder J. Singh, PhD, is the co-director of the Endocrine Laboratory in the Department of Laboratory Medicine & Pathology at the Mayo Clinic and Foundation in Rochester, MN.