A nine-year-old boy is brought into a clinic suspected of having a hypo- gonadism disorder. The physician ordered a test of the boys free testosterone after having considered this was the best option compared to testing the total testosterone. The test results came back at 25.0pg/ml reference range 0.1-3.2pg/ml flagged as high for the boy. Several months later a repeated test showed 28.0pg/ml reference range of 50-210pg/ml adult reference range flagged as high. Our paper will go into detail of how testosterone is tested, and relate it to the boy in figuring out what condition he may have.
Testosterone is the main sex hormone in males, and only a small fractioncirculates the blood in a free state. Most testosterone circulates bound to two proteins, sex hormone binding protein (SHBG), and albumin. Total testosterone levels are a combination or SHBG-bound, albumin-bound, and free testosterone. The bioavaible form (form that can bind to receptors) of testosterone includes that bound to albumin and the free fraction.
On average albumin holds 54%, SHGB 44% and free 2% so it appears that 56% of testosterone is in the bioavaible state (2).
So with this in mind our first situation concerns SHGB concentrations, which can be influenced by a variety of factors. SHGB concentrations can become decreased by obesity and testosterone treatments, while also becoming increased in aging (5). With almost half of the testosterone bound by SHGB, and with these common conditions in play, itÃÂs possibly to see how these factors could influence the free testosterone levels. In such circumstances a test of total testosterone may be warranted in the clinical decision-making of this patient.
Free testosterone has been estimated from the serum for some time by dialyzing serum across a semipermeable membrane with the testosterone that crosses in the serum believed to be the unbound portion or free. Most dialysis assays use small tracer amounts of radiolabeled testosterone added to the serum. This isotope is measured with the percentage multiplied by the total testosterone to calculate the free testosterone (3). With this method the patient is exposed to a radioactive source, an accurate measure of total testosterone is required, and is non-automated thus being time consuming. With only a small amount of testosterone able to cross the membrane .5-3% (5) measuring by dialysis itself can be problematic.
Another approach is to estimate free testosterone by measuring total testosterone and SHBG and calculate a free testosterone with an algorithm based on the law of mass action or by empirical equations. A still third approach measures bioavaible testosterone by precipitating SHBG with ammonium sulfate out of the solution and measuring the albumin-bound and free testosterone in the supernatant (5). This method may also be difficult to accomplish because the concentration of ammonium sulfate needs to be in a exact amount. With different methods to determine free testosterone how can you determine which method is the best to use for each situation?This being said Equilibrium dialysis of testosterone is considered the gold standard, but the method is cumbersome and affected greatly by dialysis conditions. Also dialysis of free testosterone disturbs the equilibrium along with impurities from the tracer (5). So what comes out of the membrane may not accurately show what is in-vivo.
Physicians tend to use the analog based method for testing free testosterone because they are easy to perform, lower cost, and can be automated. The problem with this method is that it tracks total testosterone to calculate free testosterone. And since men with low total testosterone are assumed to have low free testosterone hypogonadism is thus also assumed. More problems show up with how well this method can be trusted when calculating free testosterone, when also needing an accurate total testosterone to go along with it. The patient should also be tested with the total testosterone test to help make a better clinical diagnosis on his condition.
Based upon the manner testosterone circulates the bloodstream bound to proteins, or being free, and multiple ways of testing each that are not fail-proof. It appears as if testing for testosterone is not the easiest substrate in the body to go after.
To help rule out hypogonadism, which may occur at any age in males, an examination of the testes would be the best option. Before puberty testes usually are 1-3cm^3 in volume. During puberty, testes can grow up to 25cm^3 in size. The scrotum can be examined to see if itÃÂs completely fused. And using the Tanner method for genitalia, public hair, and axillary hair, puberty can be staged to see where the patient should be (3). With this physical being conducted along with a measure of total testosterone a physician should have a better judgment on whether or not this patient has hypogonadism or low testosterone levels.
Normal reference ranges are also crucially important in determining if a patient is healthy or diseased. The testosterone normal reference ranges are based on small samples of convenience, so not surprisingly normal reference ranges vary considerably from laboratory to laboratory. Reference ranges on average in healthy young men tend to be from 275-1000ng/dl with separate ranges for subgroups (1). Reference ranges in children are particularly complicated in that populations of children have different growth velocities, bone age, hair growth, and other factors to consider (1). Therefore itÃÂs not clear what puberty trait to link high or low testosterone to. Testosterone varying so much in a normal population also transfers down to when trying to determine a normal testosterone level for a child. With children being able to hit puberty at different ages it is hard to accurately set up any kind of reference range for children that is trustworthy. It is quite possible that the boy may be just fine at his current testosterone level, for a couple years later puberty may start to take off and end with him in the reference range.
In a study with 55 boys all of them displayed diurnal rhythm of serum testosterone before and during pubertal development. In pre-pubertal boys the testosterone concentrations at 0600-1000 hours showed the highest values. During puberty the concentration at 0600h was significantly higher than that at 1000, 1400, 1800, 2200, and 0200h. The lowest concentrations were observed from 1800-2200h (4). These diurnal rhythms are common and more severe in puberty, but they do tend to flatten out the older the child gets. Testing for testosterone in may cases may not take into account diurnal rhythms and the effect they may have on testosterone levels. If a young patient is tested early in the morning he will have a higher testosterone level then if he was tested for testosterone later in the evening. A possibility comes up as to if the boy was tested in the morning for his first test, and at night for his second test. This time change in testing may be significant along with other possible factors to have him appear to have a lower testosterone level.
To answer the question if the patientÃÂs results are low are high first off requires an understanding of the reference range for the patients age group. With no consistency of the onset of puberty, and taking diurnal cycles of testosterone into account, it may be hard to have an accurate reference range to compare a healthy or diseased child to. Multiple ways of testing each with positive or negatives add no consistency to a testing for testosterone. Without evaluation of the total testosterone along with other hormones it becomes hard to narrow down a solution.
1. Bhasin, Shalender, Anqi Zhang, and Andrea Coviello. July 2008. The impact of assay quality and reference ranges in clinical decision making in the diagnosis of androgen disorders. Steroids. 2008: 1311-13172. Kaplan, Lawrence A. Clinical Chemistry: Theory, Analysis, Correlation. Mosby, Inc.
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3. Kemp, Stephen MD PhD. Hypogonadism. Updated November 16, 2007. http://emedicine.medscape.com/article/922038-overview4. Norjavaara, Ensio and Carina Lindgren. ÃÂChanges of diurnal rhythm and levels of total and free testosterone secretion from pre to late puberty in boys: testis size of 3ml is a transition stage to puberty.ÃÂ European Journal of Endocrinology. (2004) 747-7575. Ronald S. Swerdloff and Christina Wang. ÃÂFree Testosterone Measurement by Analog Displacement Direct Assay: Old Concerns and New Evidence.ÃÂ American Association of Clinical Chemistry, Inc (2008): 54:458-460