Blood Hormone Testing Flaws
Many of my patients are interested to know if blood or saliva or urine is best for sex hormone testing.
The short story is here:
-Blood is never my preference
-Saliva is best to understand active hormone levels within the body
-Urine is best to study how the hormone is being broken down (this relates to risk)
I generally start with saliva testing, then I will sometimes add urine testing at a future date. I prefer liquid urine to dried urine testing for sex hormones.
If you are interested in the WHY, I recently wrote this up to explain why serum is likely quite flawed as a way to study sex hormones. Start with this, and I will write more on saliva and urinary testing in the coming weeks . . .
Serum Sex Hormones:
Blood testing (also referred to as serum testing, in this case) is the mainstay of modern medicine for good reasons. It’s fairly simple, quick, easy to collect, and a large array of health markers can be measured at once. While some patients have a legitimate fear of needles, most patients do quite well with this method. Moreover, for the vast majority of markers, blood testing is a valid way to assess and potentially diagnose. In fact, because it is such a solid testing medium, health care practitioners may not think twice about its validity. That is, until a review of several articles brings to question this long-held status quo.
A Large Challenge:
Sex hormones are made from cholesterol. Because blood is a watery substance, sex hormones and blood are like oil and water. They don’t mix. In order for sex hormones to travel in the blood, they need to be bound to a protein carrier.
Serum contains mostly bound sex hormones of which only a small portion (2-5%) is free or unbound. The percentage of free/unbound hormone available is slightly different for each cholesterol-based hormone. For example, greater than 95% of circulating testosterone and estrogen are bound to either sex hormone binding globulin (SHBG) or serum albumin, rendering it inactive to target tissues like the brain, bones, breast, skin, uterus, and vessels.
The unbound/active/free portion is usually what patient and doctors are curious to study, as this controls symptoms. The bound portion is quiescent or inactive and does not contribute to clinical relevancy. Therefore, an accurate means to measure this small, yet biologically active portion of serum hormones is necessary. There are two options: direct measurement or calculation.
Direct measurement:
Direct measurement of unbound hormones is rarely done outside of research settings as it is labor intensive and cost prohibitive. There are two common direct measurement methods. Both are laboratory measurements meant to mimic a naturally occurring biological process.
1. Equilibrium dialysis – the gold standard
In equilibrium dialysis a membrane is used that is permeable to salt, water, and small molecules such as estradiol. The membrane acts as a barrier, allowing the passage of specific substances. Diluted plasma is placed on one side of the dialysis membrane; on the other there is a buffered aqueous salt solution. The system is allowed to reach equilibrium, which occurs at 37 degrees Celsius, the standard temperature for many biological processes, including those in the human body. After equilibrium is reached, the concentration of estradiol in the buffered aqueous salt solution is measured. The assumption is that the measured concentration in the aqueous solution is equal to the concentration of free estradiol (unbound to proteins) in the blood.
Equilibrium dialysis is considered the "gold standard" because it aligns closely with the theoretical principles of the Law of Mass Action, a fundamental concept in chemical kinetics and thermodynamics: the rate of a chemical reaction is directly proportional to the product of the masses of the reacting substances, with each mass raised to the power that corresponds to its coefficient in the chemical equation. At best, this approach may provide a conceptual basis for the accuracy and reliability of the measurements - as it is not based on specific experimental evidence.
2. Centrifugal ultrafiltration
Unlike equilibrium dialysis, this method uses undiluted plasma, which is placed in an apparatus that allows centrifugation at a controlled temperature to separate components based on their density. The apparatus includes a semipermeable membrane located at the apex of a tube. During centrifugation, a small amount of ultra-filtrate is collected. If the ultra-filtrate is small, the equilibrium mixture of estradiol and its binding proteins is undisturbed and suggests that the ultra-filtrate is a representative sample of the unbound (free) estradiol in the plasma.
Calculation method:
This is the method used in most clinical labs because it is easy, quick, and efficient.
Calculation of free estradiol in serum:
E2 + SHBG + Alb <--> (E2*SBHG) + (E2*Alb)
KSHBG*E2 = (E2*SHBG)/(E2)(SHBG); KAlb*E2 = (E2*Alb)/(E2)(Alb)
Let X= all the E2: E2 + (E2*SHBG) + (E2*Alb)
Let Y= albumin: Alb + (E2*Alb)
Let Z= all the SHBG: SHBG = (E2*SHBG)
Then from the law of mass action the following cubic equation, given X, Y, and Z, can be solved for Free E2 (FE2).
(FE2)3 + (FE2A2)2 + FE2A1 + A0 = 0
FE2 = free estradiol
Alb = albumin
SHBG = sex hormone binding globulin
E2 = total estradiol
Ka = association constant (binding affinity)
Rosner W. Free estradiol and sex hormone-binding globulin. Steroids. 2015;99(Pt A):113-116. doi:10.1016/j.steroids.2014.08.005
In clinical settings, calculation via the Law of Mass Action (described above) is the prevalent method used to calculate free estrogen levels.
The use of the calculation method highlights the need for accurate starting levels of total E2, SHBG and albumin.
Calculation Challenges:
As Rosner (2015) points out, the original work on Mass Action, done in the 1800s, focused on chemical kinetics in simplified systems with reactants in relatively dilute, pH-buffered, aqueous solutions. However, when dealing with biological systems such as the human body, especially those involving bound particles, the assumptions based on these simplified models may not accurately describe the behavior of reaction kinetics.
This could explain some of the inconsistencies between direct measurement versus calculation of free estradiol shown in several studies.
Bikle (2020) explains, “Methods to measure free hormone levels are problematic as the free levels can be quite low, the methods require separation of bound and free that could disturb the steady state, and the means of separating bound and free are prone to error. Calculation of free levels using existing data for association constants between the hormone and its binding protein are likewise prone to error because of assumptions of linear binding models and invariant association constants, both of which are invalid.”
More Calculation Challenges:
The most utilized and accurate calculation method for free testosterone is referred to as the Vermeulen calculation. Although there are some differences in estimating free testosterone and free estradiol, the approach is basically the same.
Of note, substantially more time and effort has gone into the study of free testosterone in men versus the study of free testosterone and free estradiol in both women and children. Total testosterone concentrations in women are approximately 10-fold lower than those in men. Free testosterone concentrations are even lower, around 20-fold less than in men.
Because estrogens increase SHBG concentrations and bind with high affinity, SHBG levels in women are highly variable and can lead to variable measurements of total testosterone.
Binding Protein Measurement Issues:
SHBG can be measured using various methods. Each method has its strengths and limitations, and the choice of method may depend on factors such as precision, sensitivity, and availability. Commercial immunoassays are commonly used for measuring SHBG, and these assays may be standardized against a reference standard.
The World Health Organization (WHO) provides a standard SHBG preparation that laboratories can use for calibration and standardization purposes. However, there are inconsistencies with the WHO standard for SHBG, including a lower binding capacity, and lower SHBG-steroid association constant compared to previous standards.
Discrepancy in SHBG levels between different assay methods has been observed. Miller et al. (2004) found a 2-fold greater SHBG level with method comparisons. The reason for this discrepancy in SHBG levels is not clarified but has been suggested to be due to different forms of SHBG.
Of note, the SHBG concentration in plasma is increased by thyroid hormone, estrogenic hormones as previously mentioned, and liver disease; and is likewise decreased by obesity, androgens, prolactin, puberty, progestins, insulin and IGF-1.
Moreover, SHBG is sensitive to denaturation or breakdown.
Unsurprisingly, estimation of the proper association constants for E2-SHBG and E2-albumin are challenging. Rosner (2015) reports greater than a 3-fold difference between the highest vs. lowest association constant (Ka) for estradiol-SHBG and an almost 2-fold difference between the highest vs. lowest Ka for estradiol–albumin.
Thus, depending on the choice of Ka, calculations would reveal wide discrepancies.
But SHBG measurement is not the only challenging issue. SHBG binds 2-3x less tightly to E2 than it binds to testosterone, rendering a fixed value for albumin impossible until the appropriate calculations and experiments are determined. Finally, it is possible for a variety of other steroids to compete with SBHG and albumin for binding with estradiol, though this is rarely taken into account in calculated values. Another possible variable is the decreased affinity with albumin to estradiol and testosterone, which comes with age.
As shown in the free estradiol calculation, SHBG and albumin are the only binding agents used in the standard calculation of free estradiol and free testosterone. However, several studies suggest that there are other hormone carriers to consider, namely: the red blood cell membrane and/or lymphatics.
Even negligible variation in binding protein capacity (i.e. a 1% difference) can produce dramatic changes in reported free steroid levels. Thus, total serum estradiol is likely a poor predictor of free estradiol in women. And, as is suggested by the authors Lu (1998) and Ellison (1999), measuring salivary estradiol is likely more accurate than serum total estradiol as a predictor of free estradiol level.
Summary:
Despite all these issues, calculation is still the most prevalent way that free estradiol is reported. With consistent calculation factors, the results between serum measurements can be comparable, thus giving the false impression of “accuracy by the majority’’.
If the methods used for free hormone calculations are flawed, it naturally raises questions about the accuracy and reliability of the results and testing methodologies.
Methods shown to be inconsistent with serum values are not necessarily flawed; and methods consistent with serum values may not be inherently correct.
While measuring sex hormones in serum is economic, efficient, and simple, there remains the potential for variation and/or inaccuracy in calculation validity. When the correct association constant is an ongoing debate and the standardization is problematic, these discrepancies around SHBG binding represent an unsettled issue in laboratory science.
As Matsumoto (2004) so eloquently states “this type of work [sex hormone laboratory testing] is relatively non-sexy (despite the hormone being measured) and would be viewed as pedestrian by many funding agencies. These papers make for somewhat daunting reading because of the density of the methodological descriptions (both measurement and statistical), but the results are very important for both clinical and research audiences.”
Overall, clinicians (and their patients) need to be aware of the limitations associated with hormone measurements, even when using the perceived “gold standard” sample type - serum.
Key Points:
SHBG (sex hormone binding globulin) and albumin bind the majority of testosterone and estradiol in the blood
Free hormone is biologically active (which is approximately 2% of total estradiol, for example), while bound portions are inactive
Law of Mass Action is used for calculation free hormones (however, this is a theoretical basis not proven in biological systems/humans), with accurate total hormone, SHBG, and albumin measurements being essential (accuracies with all of these factors are problematic)
Vermeulen calculation is widely used for free testosterone determination and is considered the most accurate (this was studied in men, not in women or children and is also the basis for calculating estrogen)
SHBG concentration is affected by thyroid hormone, estrogens, liver disease, obesity, androgens, puberty, progestins, insulin, and IGF-1 (however the calculation method cannot account for any of this)
Other steroids and age-related changes in albumin affinity impact estradiol and testosterone binding and free hormone calculation (the calculation method cannot account for this)
Small variations in binding protein capacity (even as small as 1%) can significantly affect reported free steroid levels
I vote that we question the status quo and realize that, if given the choice, saliva is a preferred method to determine free/active estrogen levels, especially in women and children. Serum calculations are greatly flawed, and though efficient, there are multiple questions as to its accuracy as the “gold standard” in medicine.
-Dr. Laura Neville
References
1. Bikle DD. The Free Hormone Hypothesis: When, Why, and How to Measure the Free Hormone Levels to Assess Vitamin D, Thyroid, Sex Hormone, and Cortisol Status. JBMR Plus. 2020 Nov 2;5(1):e10418. doi: 10.1002/jbm4.10418. PMID: 33553985; PMCID: PMC7839820.
2. Rosner W. Free estradiol and sex hormone-binding globulin. Steroids. 2015;99(Pt A):113-116. doi:10.1016/j.steroids.2014.08.005
3. Érdi P; Tóth J (1989). Mathematical Models of Chemical Reactions: Theory and Applications of Deterministic and Stochastic Models. Manchester University Press. p. 3. ISBN 978-0-7190-2208-1
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