Optimal - The Blog
May 10, 2022
There are some pitfalls in measuring testosterone levels in our patients. In this post, I want to tackle the issue of which testosterone to measure (answer: Total Testosterone), which NOT to measure (Answer: Free testosterone), why it's better to calculate Free T from additional biomarkers (hint: Total Testosterone, Albumin, and SHBG), and why this provides a good measurement of Free T as well as giving us a few more biomarkers: Bioavailable Testosterone, % Free testosterone, and % Bioavailable testosterone. I hope this post is useful for you and your practice!
What is Testosterone?
Testosterone is a powerful anabolic hormone. That means it stimulates the body's development of muscle, bone, skin, and sex organs, along with masculine physical features, such as hair growth. For both men and women, testosterone helps maintain libido.
In men, testosterone is produced in the testes, by a group of cells known as Leydig cells. In women, testosterone is produced primarily by the ovaries and adrenal glands.
Peak testosterone levels are reached in a man's early to mid- 20s. As a man ages, the Leydig cells that secrete testosterone begin to wear away. Because of this, between the ages of 40 and 70, the average man loses nearly 60% of the testosterone inside his body!
Low testosterone levels in men are associated with many adverse health conditions, including diminished libido, erectile dysfunction, loss of muscle tone, increased abdominal fat, low bone density, depression, Alzheimer’s disease, and heart disease.
This is why it is essential to measure testosterone in all male patients over the age of 40.
Which Testosterone to Measure?
So now we understand some of the diseases, disorders, and dysfunctions associated with testosterone, let’s look at how we can evaluate testosterone levels in the serum. The first question I usually get is Why the serum?
The answer is that it gives us a good static measurement of blood levels and bioavailable levels in the body.
Also, because we are collecting a blood sample, we might as well order these extra few tests that can tell us a lot about not only sexual function/ dysfunction but also are part of a thorough cardiovascular risk workup.
The 2 measurements I recommend we use for assessing testosterone levels are total and free testosterone:
Total Testosterone
Total testosterone consists of all the free and unbound testosterone and all the bound testosterone.
This is not dissimilar to total T3 and free T3 in thyroid hormone evaluation.
- Testosterone circulates in the blood non-specifically bound to albumin and specifically bound to sex hormone binding globulin (SHBG).
- Less than 2% of testosterone is typically found in the “free” (uncomplexed) state in the serum of both men and women, and the remaining 98% is bound.
Measuring total testosterone is very accurate, and I recommend that you add it to all of your panels because it’s such an important biomarker to measure in our adult patients.
As you’ll see in a few paragraphs, measuring Free Testosterone is not as cut and dry as measuring Total Testosterone. You can measure it, but the units vary from lab to lab, depending on their method. My recommendation is to calculate Free testosterone, but I’m getting ahead of myself!
Why measure Total Testosterone? Because it can aid in the assessment of androgen decline and hypogonadism. In men, total testosterone (complexed and uncomplexed) is useful for assessing gonadal, adrenal, and pituitary function.
Free Testosterone
Testosterone is specifically bound to Sex Hormone Binding Globulin (SHBG) and non-specifically bound to albumin.
Roughly 54% of testosterone is bound to SHBG in men and 78% in women.
This leaves testosterone bound to albumin and free un-bound testosterone.
This has been called bioavailable testosterone, but in order to get a sense of what’s going on with testosterone, we need to measure free testosterone. Here’s where it gets complicated because there are a number of different ways of measuring or getting this value.
The 2 ways to measure Free testosterone are either by direct measurement or via calculation.
Methodologies for Measuring Free Testosterone
1. Immunochemiluminometric Assay (ICMA) followed by LC/MS/MS:
The first is via Immunochemiluminometric Assay (ICMA) followed by LC/MS/MS (Liquid Chromatography/Tandem Mass Spectrometry). Quest Diagnostics and other labs employ this two-step approach where ICMA is used for initial screening, and LC/MS/MS is utilized for confirmation and precise quantification. LC/MS/MS is considered the gold standard for testosterone measurement due to its high specificity and sensitivity. This method is probably “the physiologically” most exact method for estimating free testosterone levels. However, the accuracy of this method can be affected by the specificity of antibodies used in ICMA and the technical complexity of LC/MS/MS, requiring highly skilled laboratory personnel. In summary, it is time-consuming, technically complex, equipment-intensive, and cumbersome. Despite this, it’s the gold standard for direct free testosterone measurements. This is the method Quest uses to get us Free testosterone.
2. Direct Analog Enzyme Immunoassay (EIA)
The second is a method called Direct Analog Enzyme Immunoassay (EIA). LabCorp uses this technique, which directly measures free testosterone without requiring preliminary separation steps. While EIA is simpler and faster than LC/MS/MS, it can suffer from cross-reactivity with other steroid hormones, potentially leading to less accurate results. The performance of EIA can also vary depending on the specific assay design and the quality of the antibodies used. It is often considered the easiest and fastest method for measuring free Testosterone, but it’s not without its critics.
The problem with these direct measurements is the reference range for free testosterone, according to LabCorp using the EIA method, is about 4 times lower than Quest using ICMA and LC/MS/MS. There is no way to convert between these 2 methods.
This leaves us with our final method of determining free testosterone, which is by calculation rather than direct measurement. This is my recommended method.
3. Calculation Methods for Measuring Free Testosterone
Free testosterone can be calculated by using the following 3 biomarkers in what has been known as the "Vermulein Method" of calculating Free testosterone.:
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Total Testosterone (direct measurement by LC/MS/MS)
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SHBG
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Albumin
This approach estimates free testosterone levels based on total testosterone, sex hormone-binding globulin (SHBG), and albumin levels using algorithms derived from equilibrium dialysis measurements. While calculation methods are non-invasive and cost-effective, their accuracy can be influenced by the precision of the input values and the assumptions made by the algorithm, especially in conditions that alter SHBG levels or testosterone binding.
Without going into too much detail, the background validation of this method can be found in this article:
“J Clin Endocrinol Metab 84:3666-3672, 1999 – A critical evaluation of simple methods for the estimation of free testosterone in serum”
I mentioned above that “the physiologically” most exact method for estimating free testosterone levels was Immunochemiluminometric Assay (ICMA) followed by LC/MS/MS (Liquid Chromatography/Tandem Mass Spectrometry), the method used by Quest and many other labs (with the exception of LabCorp). Interestingly, the Vermulein method of calculating Free Testosterone is as close as you can get to getting a result consistent with Quest's method. So much so that for screening purposes I prefer to use the calculated measurement of Free testosterone over the direct measurement from Quest!
Important Caution: While calculated free and bioavailable testosterone levels provide dependable results in many clinical scenarios, they may not be accurate in situations where steroids significantly interfere with sex hormone-binding globulin (SHBG) binding. This is particularly relevant for women during pregnancy and men undergoing treatments that cause elevated levels of dihydrotestosterone (DHT), such as transdermal DHT, oral testosterone, or mesterolone therapy.
Understanding "Accuracy" in Lab Tests
In the context of laboratory tests, "accuracy" refers to how close a test's measurements are to the true value. However, this term is often conflated with related concepts such as:
- Precision (Repeatability): How consistent are the results upon repeated measurements under unchanged conditions?
- Sensitivity: The ability of the test to correctly identify those with the condition (true positive rate).
- Specificity: The ability of the test to correctly identify those without the condition (true negative rate).
Which Method is the Most Accurate?
Determining the "most accurate" method depends on the specific context and the priorities of the test (e.g., precision, sensitivity, specificity).
Immunochemiluminometric Assay (ICMA) followed by LC/MS/MS is widely regarded for its precision and specificity in measuring testosterone levels, making it a preferred method when absolute accuracy is critical. However, the calculated method and EIA offer practical advantages in terms of accessibility and cost, making them suitable for routine screening, with the understanding that confirmatory testing might be required in certain scenarios.
Conclusion
The choice between different methodologies for measuring free testosterone depends on various factors, including the purpose of the test, the population being tested, and the resources available. While Immunochemiluminometric Assay (ICMA) followed by LC/MS/MS offers high accuracy and is considered a gold standard, calculation methods, and EIA provide valuable tools for initial assessment and broader clinical applications.
Understanding the nuances of each method and the concept of accuracy in lab tests is crucial for interpreting results accurately and making informed clinical decisions.
ODX's Built-In Free Testosterone Calculator
The Vermulein Calculation isn't a simple calculation that you can do yourself so you'll have to use the ODX Free Testosterone calculator to perform the calculation.
Good News - The ODX Software Does This Calculation For You
The amazing developers over at ODX have built the Vermulein calculation into the ODX software. If you add in Albumin, SHBG, and Total Testosterone, the software will calculate the following:
- Free testosterone
- Bioavailable testosterone
- % Free testosterone
- % Bioavailable testosterone
By NOT measuring Free testosterone and simply substituting it with SHBG, you get 4 calculated biomarkers that will help you with your sex hormone assessments. A good trade-off, I think!
BONUS - 5 Reasons Free testosterone May Be Low
- Too much testosterone being converted to estradiol
- Too much free testosterone bound to SHBG
- Low LH secretion from pituitary
- Low output from testes
- Inadequate amounts of DHEA
