Testosterone is the main male sex steroid produced by Leydig cells in the testes, with an average secretion rate of 7 mg/day. Based on calculations from spermatic vein/peripheral vein gradients.
Leydig cells can also release intermediate metabolites such as androsterone, androstenedione, 17-OH progesterone, progesterone, and Pregnenolone.
Approximately 5% of the T pool is of adrenal derivation. Studies in patients with prostate cancer demonstrated that human adrenals produce approximately 200 μg of T regardless of whether the patient had intact testes or was castrated, and an additional 200 μg of T is formed in the periphery from the conversion of adrenal-derived androstenedion].
Plasma T is bound to the sex-hormone binding protein (~44%) and albumin (~54%), and only 2% circulates freely. These three fractions of T are measured together, as “total Testosterone”, which is a reliable frontline tool for detecting hypogonadism.
Three types of testosterone
There are three types of testosterone in the bloodstream
- SHBG bound testosterone. This is testosterone that is bound to a protein called SHBG.
- The testosterone is bound to a protein called albumin which is manufactured in the liver.
- Free testosterone, which is not attached to any proteins. This sort is the most useful for carrying out testosterone’s functions.
Total Testosterone = SHBG + Albumin + Free Testosterone
Remember, several conditions alter the absolute level of plasma SHBG, and will be associated with an increased (or decreased) serum level of total T.
SHBG (and total Testosterone) decreases in patients affected by obesity, T2DM, hypothyroidism, and nephrotic syndrome and increase with aging, pregnancy, hyperthyroidism HIV, and cirrhosis.
The level of testosterone in the blood varies during the day.
The highest levels are in the morning and the lowest levels are at night.
SHBG-bound T is not bioavailable because of the tight interaction existing between the two, which prevents SHBG-bound T to reach Androgen Receptors in the target cell.
Free and albumin-bound T represents the bioactive fraction of T. The fraction of T that enters the target cell and interacts with the Androgen Receptors.
The level of testosterone in the blood varies during the day. The highest levels are in the morning and the lowest levels are at night.
Total Testosterone test
Total testosterone is the mainstay of the biochemical diagnosis of androgen deficiency and is recommended as the initial diagnostic test.
Indeed, in an international survey among 943 mostly adult endocrinologists, more than 90% of participants requested total testosterone, drawn in the morning as the initial diagnostic for workup of suspected androgen deficiency.
In practice, a normal fasting early morning total testosterone level (somewhat arbitrarily defined as ≥12 nmol/L) usually does not need to be repeated.
If the total testosterone is ≥12 nmol/L, non-specific symptoms will generally not be from androgen deficiency unless SHBG is markedly elevated or if there is androgen resistance, a rare condition.
Low total testosterone needs confirmation (additional test) because a falsely low level due to, for example, unrecognized intercurrent illness or assay imprecision at the lower range, particularly if measured with immunoassay (see below) is more likely than a falsely normal level.
Diagnosis of androgen deficiency should never be based on a single low testosterone level. Up to 35% of men with a low testosterone level will have a normal testosterone level on repeat testing
Why you need to check free testosterone
Circulating testosterone is mainly plasma protein bound, 60% tightly to sex hormone-binding globulin, and 38% loosely to albumin, while 0.5–2% circulates as free testosterone.
A free testosterone test may be helpful when total testosterone is borderline and/or the clinical picture does not agree with the total testosterone measurement.
For example, free testosterone can be useful to exclude hypogonadism in men where low total testosterone is due to low SHBG because of insulin resistance in obesity or diabetes.
In this context, normal free testosterone can be reassuring in that nonspecific symptoms are not due to androgen deficiency. However, the age-related decline of free testosterone is steeper than that of total testosterone because of the age-associated increase in SHBG.
A low free testosterone level should be evaluated with caution to confirm hypogonadism in older men
because the risk of overdiagnosis is substantial given that assay reference ranges are usually based on findings in young men.
An extreme case of a “falsely low” total testosterone was recently described in a man who presented with an extremely low total testosterone level but essentially normal masculinization. SHBG levels were undetectable because of a missense mutation in the SHBG gene, and dialyzable free testosterone was in the reference range.
In certain instances, men can be androgen deficient despite a normal total testosterone level, and this usually occurs if SHBG is markedly elevated most commonly in the setting of anti-epileptic treatment or chronic liver disease, but these men typically have elevated gonadotropin levels and a clearly low free testosterone level.
Free Testosterone Test Methods
Laboratory equilibrium dialysis is the gold standard for free testosterone measurement, but is not widely available, because of assay complexity and cost.
The free androgen index is inaccurate in men and should not be used, and free analog displacement using direct free testosterone (analog) assay is analytically invalid and should not be used.
In practice, free testosterone is usually calculated using empiric formulas.
Five different formulas (two based on equilibrium binding, three empirical) are commonly used to calculate free testosterone.
There is currently no universally accepted formula that accurately reflects the interaction between plasma protein-bound and free testosterone.
In addition, these formulas are critically dependent (80 % variance) on the accuracy of the total testosterone and SHBG assays, and may augment errors in their measurement.
During hypogonadism with aging, testosterone falls from maximal concentrations to the end decade of life.
Free testosterone falls faster than total testosterone.
Recent data indicate that testosterone replacement may enhance sexual drive and function in older men.
- Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982;16:801–10.
- Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84:3666–72.
- Ly LP, Sartorius G, Hull L, Leung A, Swerdloff RS, Wang C, Handelsman DJ. Accuracy of calculated free testosterone formulae in men. Clin Endocrinol (Oxf). 2010;73:382–8.
- Raivio T, Palvimo JJ, Dunkel L, Wickman S, Janne OA. A novel assay for determination of androgen bioactivity in human serum. J Clin Endocrinol Metab. 2001;86:1539–44.
- Jarow JP, Troiani J, McNellis D, Wiederhorn R, Fang G, Handelsman H. Use of biomarkers to assess tissue-specific androgen adequacy: defining male hypogonadism. J Urol. 2013;189: 633–7.
- Wang C, Nieschlag E, Swerdloff R, Behre HM, Hellstrom WJ, Gooren LJ, Kaufman JM, Legros JJ, Lunenfeld B, Morales A, Morley JE, Schulman C, Thompson IM, Weidner W, Wu FC. Investigation, treatment, and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA, and ASA recommendations. J Androl. 2009;30:1–9.
- Bhasin S, Pencina M, Jasuja GK, Travison TG, Coviello A, Orwoll E, Wang PY, Nielson C, Wu F, Tajar A, Labrie F, Vesper H, Zhang A, Ulloor J, Singh R, D’Agostino R, Vasan RS. Reference ranges for testosterone in men were generated using liquid chromatography-tandem mass spectrometry in a community-based sample of healthy nonobese young men in the Framingham Heart Study and applied to three geographically distinct cohorts. J Clin Endocrinol Metab. 2011;96:2430–9.
- Yeap BB, Alfonso H, Chubb SA, Handelsman DJ, Hankey GJ, Norman PE, Flicker L. Reference ranges and determinants of testosterone, dihydrotestosterone, and estradiol levels measured using liquid chromatography-tandem mass spectrometry in a population-based cohort of older men. J Clin Endocrinol Metab. 2012;97:4030–9.