CLN’S

LAB 2006:
SUPPORTING
CLINICAL
DECISIONS
SERIES

Testosterone

Total, Free, or Bioavailable: What Should Labs Report?
BY RAYMOND LEPAGE, PHD

T

raditionally, society has viewed menopause as something that happens only to women, but today
the concept of male menopause is receiving greater attention by both the medical community
and the general public. Researchers first described symptoms associated with male menopause
in the 1940s, but the concept did not became widely recognized until the mid-1990s, when aging
baby boomers were invited to attend well organized “andropause clinics” supported by the pharmaceutical industry. Today, many men use pharmaceutical testosterone to help with lowered sex drive, loss of
energy, and other symptoms associated with middle age.
In addition to being a common effect of aging, testosterone deficiency can result from a variety of other
causes. In men with true hypogonadism, androgen replacement improves sexual functioning and maintains
bone density and lean body mass. In past years, however, the preferred method of androgen supplementation
for these men involved extremely unpleasant scrotal patches, which ultimately restricted treatment to patients
with overt primary or secondary hypogonadism. More practical ways of administering testosterone have since
been introduced, with non-scrotal transdermal patches and oral preparations now providing less painful treatment options. Although oral testosterone preparations are not available in the U.S., the Food and Drug Administration has approved a transdermal testosterone gel for treatment of hypogonadism.
With these new advances, clinicians have
also gained more acceptable forms of testosterone supplementation for the treatment of
andropause, a condition best described as
partial androgen deficiency of the aging male
(PADAM) that is thought to affect more

include a complete medical history and hormonal screening. To support diagnosis and
treatment of testosterone deficiencies, clinicians therefore need an accurate measurement of testosterone levels, but providing
high quality testosterone results has been a

cells. After it is produced in the cytoplasm of
the Leydig cells, testosterone readily crosses
the nuclear membrane and binds either as
testosterone or as dihydrotestosterone to
a protein receptor. This receptor complex
exerts its effects mostly on gene expression,
producing the familiar male characteristics.
In blood, approximately 50% of testosterone is tightly bound to a liver protein called
sex hormone binding globulin (SHBG). The
other half binds loosely to albumin, leaving
only approximately 2% as free testosterone.
For many years, researchers believed that
all the biological actions of testosterone,
including the secretion of luteinizing hormone (LH), were under the control of this
small free fraction of the hormone.
Today, the fraction of testosterone in
blood that is bound to albumin is widely, although by no means universally, believed to
be like free testosterone, in that it is available
for tissue uptake and therefore biologically
active (see box, p. 14). Therefore the fraction
of hormone comprising free testosterone
plus that bound to albumin—or the nonSHBG-bound fraction—is termed “bioavailable” testosterone.

Methods for Measuring the
Subfractions of Testosterone

than 5 million American males (see upper
box, p. 13). Clinicians diagnose PADAM
based on the observation of a number of
rather nonspecific symptoms accompanied
by a lower blood testosterone level than that
measured in young adult males.
Considering the significant risks of testosterone supplementation—prostate cancer, increased hematocrit, and cardiovascular risks—evaluation of potential candidates
for testosterone replacement therapy should

12

CLINICAL LABORATORY NEWS AUGUST 2006

real challenge for labs. This article describes
the controversy surrounding the measurement of total, free, and bioavailable testosterone, as well as how labs can address some
of these issues.

From Free Testosterone to
Bioavailable Testosterone
Although a small amount of testosterone is
produced in the adrenals, males produce testosterone primarily in the testicular Leydig

As mentioned earlier, about half of the testosterone in blood circulates tightly bound
to SHBG. Numerous conditions, many of
which are independent of total testosterone,
affect the level of SHBG (see lower box, p.
13) and consequently the measured level of
total testosterone. The amount of the biologically active fraction, whether free or bioavailable, readjusts rapidly via modified secretion
of pituitary LH. This situation is identical to
the relationship between total thyroxine,
free thyroxine, and TSH. Similar to total T4,
total testosterone does not always reflect the
quantity of active hormone, and in many
cases measured levels of total testosterone
must be adjusted for variations in SHBG. As

But as the seemingly endless debate about whether clinicians should use free or bioavailable testosterone measurements to manage patients continues. however. originally proposed in 1989 by Déchaud. the operator first adds a tracer dose of 3H-testosterone to a serum sample and allows it to distribute between the free fraction and the fraction bound to proteins. and the index does not take into consideration the influence of albumin and other proteins to which testosterone binds. but this ap- amount of SHBG-bound testosterone from total testosterone. Measuring bioavailable testosterone by precipitation. To assess the amount of bioavailable testosterone. The two other approaches for evaluating free testosterone levels.issam. Moving Past the Debate While supporters of the free hormone theory say that bioavailable testosterone is a surrogate marker.) (8). SHBG readily precipitates in the resulting halfsaturated solution. With the exception of the direct free analog assay. The other assay that has been widely available in North America to measure free testosterone is the “direct” free testosterone assay. free analog assays for testosterone have been regularly criticized in the literature. stochiometric equations show that under conditions of slight variation of the albumin level. is much more difficult. More than 30 years ago. this method has also been highly criticized in the literature.htm Bioavailable Testosterone Measured by the Fraction of Nonprecipitable Testosterone. directly measures the testosterone in the supernatant (6). Because of these shortcomings. In order to obtain clinically valid evaluations of free testosterone levels. All of these steps are performed at 37°C to represent the actual equilibrium conditions in the body. In this approach. most likely lies elsewhere.ch/freetesto. proach has not been very successful. a piece of equipment that is not readily available in most clinical laboratories. As with all surrogate markers. such as in PADAM subjects. Only two research groups have proposed direct approaches: a coatedtube. the method is quite labor intensive. which basically limits the use of the assay to female patients. is hampered by numerous technical difficulties. however. The real problem in providing reliable testosterone results. and the operator uses a liquid scintillation counter to measure the fraction of tracer in the dialysate or the ultrafiltrate. the major one being obtaining good precision in the precipitation step. For supporters of the theory of bioavailable hormones. While the same operator can easily attain good within-assay precision on duplicate specimens in small batches. which must be measured independently. and very few clinical laboratories currently measure free testosterone in this manner. laboratorians still need to provide test results. In addition. Measurement of SHBG and Calculation of a Free Androgen Index (FAI). N.Y. Stochiometric Calculation of Free Testosterone.Signs and Symptoms of Andropause ® Loss of libido ® Erectile dysfunction ® Depression ® Lethargy ® Inability to concentrate ® Sleep disturbance ® Irritability ® Depressed mood ® Osteoporosis ® Loss of muscle mass and strength described below. Not only does this assay require extensive expertise and time. manual assay (7). To arrive at the level of bioavailable testosterone. Researchers have proposed values for this affinity constant that differ by as much as a factor of four. Tremblay and Dubé (5) proposed the use of a tracer dose of 3H-testosterone added to the specimen and the calculation of the percentage of the tracer that remains in the supernatant. has recently been gaining popularity in North America. and in fact results obtained by these assays correlate rather poorly with free testosterone measured by dialysis. both require the measurement of the less popular SHBG protein. Some researchers have also attempted to use an immunoassay to directly measure the non-precipitated testosterone in the ammonium sulfate supernatant. Measurement of Free Testosterone by Dialysis or Ultrafiltration. In addition. laboratorians must understand that the assay uses antibodies with low affinity relative to SHBG (Ka = 1×109 L/M). this approach has several limitations. particularly with changing personnel. The other approach. Based on the same principle as the measurement of free thyroxine by an analog or one-step assay. while albumin-bound and free testosterone remain in solution. leading experts in testosterone have suggested that liquid chromatography/tandem mass spectrometry (LC/MS/MS) may be a much better alterna- CLINICAL LABORATORY NEWS AUGUST 2006 13 . Direct Measurement of Free Testosterone by an Analog-type Competitive Immunoassay. Moreover. the use of a radio-labeled tracer makes it expensive. judging from the very large inter-laboratory variation in external proficiency surveys—in excess of 40%—this assay hardly lives up to its “gold standard” label. with the results of direct free testosterone assays being one order of magnitude lower than those obtained by dialysis. obtaining good between-assay CVs. between free testosterone (as FAI) and total testosterone. rather than a measured value. although it does not exist. the choice of using one value versus another for the affinity constant could have a greater impact on the calculated free or bioavailable testosterone result than using a constant. However. a method that was first introduced in Europe. Imagining that these “low” affinity capture antibodies will not displace weakly bound testosterone from albumin (Ka = 1×104 L/M) might seem difficult. they then subtracted the Equation for the Calculation of Free and Bioavailable Testosterone TT = Ks * [SHBG] * FT 1 + (Ks * FT) + Ka * [Alb] * FT + FT 1 + (Ka * FT) TT = Total testosterone (mol/L) Ks = Association constant for T-SHBG binding (L/mol) Ka = Association constant for T-Albumin binding (L/mol) SBHG = SHBG concentration (mol/L) FT = Free testosterone (mol/L) Alb = Albumin concentration (mol/L) Online calculator at: http://www. Another difficulty for labs that want to use the 3H-testosterone method is the need for a liquid scintillation counter. the concentration of free testosterone is proportional to that of albumin-bound testosterone. this free testosterone assay uses a tracer that is a testosterone analog. As automated testosterone and SHBG measurements become more readily available. the clinical information provided by either assay will be equivalent. The sample is then placed in a small dialysis bag or in an ultrafiltration device. the chemical structure of which has not been revealed by the manufacturer. This index assumes a linear relationship. Manufacturers of direct free testosterone assays have indirectly acknowledged these concerns by limiting the intended use of the assay to the “clinical diagnosis and management of hirsutism due to hyperandrogenism”. given these issues. Traditionally considered the gold standard of free testosterone measurement.” but for the vast majority of cases however. the purity of the tracer is of the utmost importance because the dialyzable fraction represents less than 2% of the dose of added tracer. To understand the assay’s results. supporters of the bioavailability of steroid hormones say free testosterone should be considered the surrogate marker. This surprising conclusion can be drawn from the fact that researchers have validated all bioavailable testosterone assays against the gold standard dialysis method of testosterone measurement. Furthermore. the percentage is then multiplied by the level of total testosterone. most likely because of the salt’s interference with the immunoassay. and an assay on the Bayer Immuno 1 Immunoassay Analyzer (Tarrytown. the tracer competes with free testosterone for binding sites on the antibody used in the assay but does not displace the equilibrium between free testosterone and albumin—or SHBG-bound testosterone. and many scientific and medical societies have asked that they be retired from the market. Researchers have proposed two approaches to complete the measurement. The FAI is a unitless measurement obtained by dividing the level of total testosterone by that of SHBG. the dialysis method of measuring testosterone is the method against which all others have been validated. and the researchers ultimately resorted to measuring SHBG-bound testosterone in the redissolved precipitation pellet. Vermeulen has proposed an equation that incorporates the level of serum albumin in addition to total testosterone and SHBG Conditions Affecting SHBG Levels in Males Increased SHBG ® Aging ® Hyperthyroidism ® Hypogonadism ® Alcohol cirrhosis Decreased SHBG ® Obesity ® Hypothyroidism ® Acromegaly ® Androgen therapy levels (see box below). The latter approach showed considerable interference from the ammonium sulfate. the value of the association constant for T-SHBG used in the equation (Ks) has been quite controversial (4). FAI and stochiometric calculation. researchers are uncertain whether the affinity constant varies with age. Not surprisingly. the operator mixes equal volumes of serum and saturated ammonium sulfate at 4°C or 37°C. In this method. Under well-defined conditions. approaches for the measurement of free or bioavailable testosterone are based on measuring total testosterone using one of the commercially available manual or automated immunoassays. therefore. To measure free testosterone. Despite being considered the gold standard method for measuring free testosterone. nothing beats the actual measurement of testosterone that is not bound to SHBG. there are several ways that laboratories can make this adjustment. stochiometric calculation of free testosterone. In fact. for albumin. Recent editorials in major endocrine journals criticized the quality of testosterone immunoassays (9). there will be rare cases where no test will replace the “real thing. and the method is not amenable to automation.

390+ pages. Editorial: Serum testosterone assays—Accuracy matters. et al. On the other hand. I recommend that laboratorians focus on the quality of their current CLN total testosterone assay. Empiric testing of the free hormone hypothesis. Clin Chem 1989. Contraception 1974. free testosterone. 35: 1609–1614. While LC/ MS/MS methods are less common in clinical labs. Click on “Publications. calibration material is theoretically impossible to define. A More Targeted Approach to Testosterone Measurements In addition to providing better clinical information. the dogma was not seriously contested until the mid1980s when Pardridge conducted the first-pass kinetic experiments (1). and eventually this idea evolved into a dogma that was applied to all small hormones. Vermeulen A. Clin Chem 2002. REFERENCES 1. His hypothesis was based on the presence of undefined mechanisms in the microcirculation that were capable of enhancing the dissociation of a ligand. Interestingly. such as significantly reducing the need to measure the various subfractions of testosterone. Massé J. Tremblay RR. Tremblay RR. et al. Dubé JY. it nevertheless gained credibility and began to appear in major endocrine textbooks. In such extreme cases. 58 pages. gouv. Making a Choice With so many methods and so many drawbacks. some researchers question whether the equation applies equally to all individuals. laboratorians have struggled with choosing whether to report results of calculated free and bioavailable testosterone or precipitated bioavailable testosterone. In some tissues.org Phone: Call AACC (800) 892-1400 or (202) 857-0717 14 CLINICAL LABORATORY NEWS AUGUST 2006 . Giton F. softcover ISBN 9781594250477 Product # 2450 Principles of Forensic Toxicology. 74: 745–752. J Clin Invest 1984. 38: 813–818. from low-affinity binding proteins like albumin. walk-away equipment now offers clinical labs the means to provide highly accurate and precise testosterone results. esis: A physiologically based mathematical model. Catania 2006. even tightly bound ligands could dissociate from high-affinity carriers like SHBG. 4. 48: 388–390.ca. the use of a reflex testing algorithm could result in substantial economies by reducing the number of assays a lab performs to measure the subfractions of total testosterone (10). J Clin Endocrinol Metab 2004.” New Titles from An A–Z Guide to Pharmacogenomics Michael G. as well as the substantial economies that could be realized by measuring only total testosterone. such as testosterone or thyroxine.aacc. 84: 3666–3672. or bioavailable testosterone. in order to provide high quality testosterone results to clinicians. Bound Hormones Which is the Biologically Active Form? The concept that only free hormones are biologically active originated from studies of thyroid hormones in the 1950s. Therefore. et al. J Clin Endocrinol Metab 1999. 2. a total testosterone level of 58 ng/dL (2 nmol/L) cannot be accompanied by a higher. Serum bioavailable testosterone: Assayed or calculated? Clin Chem 2006.lepage. including testosterone. For example. 8. 10: 232–274.aacc. When is bioavailable testosterone a redundant test in men? Clin Biochem 2005. Except in very rare cases. the concept of “bioavailable thyroxine” has never been adopted. Email: raymond. Endocr Rev 1989.tive to traditional immunoassays. Déchaud H.” “Clinical Laboratory News. PhD. Raymond Lepage. although the Pardridge theory applies to all small hormones and drugs. Gheorghiu I. any other measurement will yield the same clinical information. While the basic assays used to report calculated values are more easily controlled. there is no additional information that can be obtained from measuring SHBG. Considering the drawback of both approaches present here. 7. This article is available as an 8 1/2” x 11” reprint on the AACC Web site (www.qc. et al. 2: 16–21. 6. Landaw EM. Matsumoto AM. Aging Male 1999. Furthermore. Davies R. Indirect measurement of bioavailable testosterone with the Bayer Immuno I system. A critical evaluation of simple methods for the estimation of free testosterone in serum. is Clinical Biochemist in charge of technologies in the Clinical Biochemistry Department of the University of Montreal Medical Center (CHUM) and Professor of Biochemistry at the University of Montreal in Quebec. Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Verdonck L. The same principle applies for high total testosterone levels. Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma.chum@ssss. many researchers have limited the use of these three assays to specimens in which the total testosterone ranges from 170 to 400 ng/dL (6 to 14 nmol/L). Revised and Updated Second Edition Edited by Barry Levine 2006. 10. easy-to-use. Usefulness and limitation of bioavailable testosterone in assessment of androgenicity during the process of aging in men. 52: (In press). normal level of the bioavailable testosterone subfraction (> 85 ng/dL or >3 nmol/L). Kaufman JM. Although researchers discovered in the early 1960s that the free hormone concept could not explain all the data on tissue uptake of small hormones. 10: 599–605. if a total testosterone is outside this range. Pardridge WM. and quality control material is scarce. Mendel CM. Bremner WJ. Plasma concentrations of free and non-TeBG bound testosterone in women on oral contraception. softcover ISBN 9781594250538 Product # 4202 Two Easy Ways to Order! Online: www. 9. 5. the precipitated approach is a homebrew assay for which there is no reference method.” then “Series Articles. 3. 89: 520–524. Although this new theory was vigorously contested in the late 1980s (2). the recent introduction of automated.org). putting more emphasis on providing accurate total testosterone measurements could have additional benefits for labs. The free hormone hypoth- Free vs.