For Care Providers

Methods for the Determination of Steroid Hormones

Choice of Specimen

For many years, very few methods were sensitive enough to measure steroids in blood; therefore, urinary measurements were widely used. However, because of a number of technical developments, methods are now available that allow steroids to also be measured in blood and saliva.

Urine
Although the urinary excretion of a hormone, of its metabolites, or of both, does not account for the total amount of hormone secreted by the gland, it usually represents an approximate proportion of the amount secreted during the period of urine collection. Thus, urinary assays are considered to reflect the secretory activity of the endocrine glands. However, factors such as incompleteness of collection, altered renal function, and contribution by more than one gland to the total excretion of the same hormone or hormones (e.g., adrenal glands and gonads both contribute to the level of androstenedione) warrant special attention in the interpretation of urinary values. Analyses of total urinary metabolites have further shortcomings because they reflect only a fraction of the active steroid hormones that are metabolized through different pathways; the quantity and the nature of these metabolites may, in turn, depend not only on the pathological condition but on the intake of drugs and on diet. Other arguments against urinary assay of metabolites include the time and inconvenience encountered in the collection of 24-h urine specimens. Urine assays, however, do have their place in the determination of free hormone. Urinary free cortisol and the measurement of urinary free estradiol, estrone, and testosterone have been shown to provide clinical information that can reflect the production rates of these steroids.

Blood
The determination of steroids in blood has now largely replaced urine assays for routine purposes and is much more convenient both for the laboratory and the patient. Immunoassays and chromatographic methods have been developed that can efficiently determine virtually all of the clinically relevant steroids found in the circulation. In addition, because endocrine testing is founded on provocative testing (stimulation and suppression tests), blood samples for rapid dynamic testing are much more appropriate than urine samples. Plasma determinations, however, still have their limitations because of the rapid fluctuations and pulsatility that occur in hormonr levels: thus, a plasma sample is representative only of the concentration that existed at the time of the sampling. Therefore, the measurement of some steroids, such as free cortisol, using timed urine collections still has value.

There is no experimental evidence that steroid hormone levels in serum are different from those in plasma. However, rapid separation of red blood cells in the specimen is important because red blood cells at room temperature can alter plasma concentrations of active steroid hormones; red blood ceels degrade estradiol to estrone and cortisol to cortisone, and they can adsorb testosterone.

Saliva
In general, most steroids of clinical interest can be measured in saliva.¹⁹¹ For some steroids, such as cortisol, estriol, and progesterone, the measurement of the salivary level appears to be a reliable indicator of the free concentration in plasma. For others (e.g. testosterone, 17-hydroxyprogesterone, estradiol, and aldosterone), the clinical usefulness of salivary measurements has not yet been fully established.

It has been suggested that measurement of salivary steroids reflects the free (nonprotein-bound) steroid fraction in blood and may provide information similar to that derived from measurement of urinary free steroids.^139.148 The measurement of both cortisol and progesterone have been determined in saliva. Salivary sampling protocols are advantageous in that they make for frequent and easy collection of samples by noninvasive, stress-free techniques. Moreover, they obviate the difficulties of ensuring the completeness of a 24-h urine collection. Patients find little difficulty in salivating directly into disposable tubes and can provide an adequate volume in ~10 min. Assays of samples collected at 1- to 2-h intervals during waking hours provide an accurate assessment of baseline endocrine activity. Because smaller aliquots can be collected at 15- or even 10-min intervals, salivary samples could well be more useful than either plasma or urine samples with short-term dynamic testing protocols. The possibilities and pitfalls of salivary hormone assays have been further discussed by Vining and McGinley.¹⁹¹

Excerpt from Tietz Textbook of Clinical Chemistry, 3rd Edition, 1999

ADDITIONAL INFORMATION
Pre/Post Adrenal Stimulation	Adrenal	Thyroid Panel	Serum DHEA
Methods for the Determination of Steroid Hormones