Calculate Free Testosterone with TruT by FPT

[tareload]

Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms

To develop and validate liquid chromatography tandem mass spectrometry (LC–MS/MS) methods for the direct measurement of total and free testosterone in…

www.sciencedirect.com

See method validation and method comparison sections:

See panel E:

 

[sammmy]

Reference lab ranges are specific to the actual test used and they just tell you the relative position of the given specimen with respect to the group of people whose statistical data was used in constructing the reference range.

Reference ranges of free testosterone cannot be compared between different lab tests because they do not actually measure the absolute value of free testosterone but some proxy of it, depending on the test.

The best one can do is select one of the tests or a calculator that are considered consistent and see how the personal values change over time on the same test or calculator. The reference range will also tell you if you are 'high' or 'low'. Trying to compare values between different tests or calculators is simply a waste of time.

 

[xqfq]

In conclusion, I can't find one reference laboratory in the US that has a equilibrium dialysis free T reference range for Adult males that is anywhere near 16-31 ng/dL.

Equilbrium Dialysis Reference Ranges for free T (ng/dL) - Adult Male

1. Quest 3.5-15.5

2. LabCorp (Endocrine Sciences) 5.2-28

3. Mayo 3.7-14.5 (mean of sliding scales)
low (2.3-5) - high (8-21) sliding scale range by age group

4. ARUP 4.7-24.4

5. University Hospital Ghent 3.4 - 12.3

reference range from this paper:

Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis

Reassessment of FT in women and men with state-of-the-art methodology confirms previously established FT percentages but highlights limitations of differen

academic.oup.com

View attachment 11220


So which reference laboratory has a reference range for their free T by ED anywhere near 16-31 ng/dL?

@madman @Cataceous @Nelson Vergel @Dr Justin Saya MD @sammmy @DS3

Based on the quote from madman:

"Based on the new data on the distribution of free testosterone levels in healthy men the target range of free testosterone has been determined to be 164 to 314 pg/ml (mean+/−1SD)"

I took the 'range' to not mean a 'range of a random selection of blood samples' but rather an effort to identify the 'therapeutic range' for healthy men's free T level[1]. Unfortunately, I cannot find a source for this quote, though I did find some information in a patent filing:

WO2014138026A1 - Methods and systems for the diagnosis and treatment of androgen disorders - Google Patents

The technology described herein is directed to the diagnosis and treatment of androgen disorders and/or deficiencies, e.g, low testosterone.

patents.google.com

I remember reading something more concrete on this 'range' a while ago, but I can't find it now.

1. To the best of my knowledge, there is no published paper on such a therapeutic range, though it would be very valuable. The 'healthy non-obese' subjects used in the latest labcorp range are majority-prediabetic and majority-prehypertensive, for instance. I also have concerns about a BMI shift over time, wherein the average fat mass of a male now is likely higher than it was, say, 30 years ago. The study which labcorp draws its range from (Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe) does not 'adjust' for this likely BMI shift, but instead simply excludes anyone with BMI >= 30. A study which also measured things like energy levels, libido, mood / depression, would be much more valuable. Instead, the range likely reflects an increasingly sick population.

On this subject,

is an interesting discussion about age-related decline and when to initiate therapy.

 

[tareload]

Reference ranges of free testosterone cannot be compared between different lab tests because they do not actually measure the absolute value of free testosterone but some proxy of it, depending on the test.

Equilibrium dialysis followed by LCMS/MS is absolutely a free testosterone assay that measures the absolute value of free testosterone. Read the calibration standardization any lab has to do qualify the test.

If what you say is correct, this graph would not exist:

Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms

To develop and validate liquid chromatography tandem mass spectrometry (LC–MS/MS) methods for the direct measurement of total and free testosterone in…

www.sciencedirect.com

If equilibrium dialysis followed by LCMS doesn't measure the absolute value of free T, then alot of people have wasted a lot of time trying to create models to fit the data. The issue I have brought up here is the disconnect between the data the authors used to create the tru-T model vs ALL the other equilbrium dialysis data I can find and all the other reference labs reference ranges. This whole thread would be pointless if each lab doing equilibrium dialysis were not attempting to measure the actual free T in serum.

 

[tareload]

Based on the quote from madman:

"Based on the new data on the distribution of free testosterone levels in healthy men the target range of free testosterone has been determined to be 164 to 314 pg/ml (mean+/−1SD)"

I took the 'range' to not mean a 'range of a random selection of blood samples' but rather an effort to identify the 'therapeutic range' for healthy men's free T level[1]. Unfortunately, I cannot find a source for this quote, though I did find some information in a patent filing:

WO2014138026A1 - Methods and systems for the diagnosis and treatment of androgen disorders - Google Patents

The technology described herein is directed to the diagnosis and treatment of androgen disorders and/or deficiencies, e.g, low testosterone.

patents.google.com

View attachment 11233

I remember reading something more concrete on this 'range' a while ago, but I can't find it now.

1. To the best of my knowledge, there is no published paper on such a therapeutic range, though it would be very valuable. The 'healthy non-obese' subjects used in the latest labcorp range are majority-prediabetic and majority-prehypertensive, for instance. I also have concerns about a BMI shift over time, wherein the average fat mass of a male now is likely higher than it was, say, 30 years ago. The study which labcorp draws its range from (Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe) does not 'adjust' for this likely BMI shift, but instead simply excludes anyone with BMI >= 30. A study which also measured things like energy levels, libido, mood / depression, would be much more valuable. Instead, the range likely reflects an increasingly sick population.

On this subject,

is an interesting discussion about age-related decline and when to initiate therapy.

That is exactly the range they measure (see this post):

Calculate Free Testosterone with TruT by FPT

[0026] Fig. 7 depicts the thermodynamic Parameters associated with testosterone's binding to SHBG derived from the fit of binding isotherms and ITC data to new model. While this parameter set is not unique, together they consistently describe the binding isotherms, depletion curves and ITC data...

www.excelmale.com

 

[madman]

In conclusion, I can't find one reference laboratory in the US that has a equilibrium dialysis free T reference range for Adult males that is anywhere near 16-31 ng/dL.

Equilbrium Dialysis Reference Ranges for free T (ng/dL) - Adult Male

1. Quest 3.5-15.5

2. LabCorp (Endocrine Sciences) 5.2-28

3. Mayo 3.7-14.5 (mean of sliding scales)
low (2.3-5) - high (8-21) sliding scale range by age group

4. ARUP 4.7-24.4

5. University Hospital Ghent 3.4 - 12.3

reference range from this paper:

Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis

Reassessment of FT in women and men with state-of-the-art methodology confirms previously established FT percentages but highlights limitations of differen

academic.oup.com

View attachment 11220


So which reference laboratory has a reference range for their free T by ED anywhere near 16-31 ng/dL?

@madman @Cataceous @Nelson Vergel @Dr Justin Saya MD @sammmy @DS3

TruTTM

Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis (2018) Assessment of FT might improve precision in the diagnosis of hyperandrogenism in women and hypogonadism in men, in particular when total T levels are borderline and in...

www.excelmale.com

 

[tareload]

It does seem like a significant discrepancy. Maybe we should just revert to using one of the empirical formulas mentioned in your other post:

Reassessing Free-Testosterone Calculation by Liquid Chromatography–Tandem Mass Spectrometry Direct Equilibrium Dialysis

Context: Assessment of free testosterone (FT) might help evaluate androgen status in patients with borderline total testosterone (T) and/or altered sex hormone–binding globulin (SHBG) levels. However, the validity of different methods to measure FT is debatable. Methods: Serum from 183 women...

www.excelmale.com

I looked up the one from Sartorius et al. It is straightforward:

cFT (pMol/L) = 24.00314 * T / log10(SHBG) - 0.04599 * T * T
T and SHBG in nMol/L
T(ng/dL) = 28.8184 * T(nMol/L)
FT(ng/dL) = FT(pMol/L) / 1000 * 28.8184

One nice thing is that the numbers somewhat align with Quest's equilibrium dialysis test, which has a reference range of 3.5-15.5 ng/dL. Using an SHBG of 30 nMol/L has this reference range corresponding to total testosterone ranging from just over 200 ng/dL to just under 1,100 ng/dL. Is it a coincidence that this corresponds to Quest's reference range for LC/MS total testosterone?

FTA and FTZ look pretty darn good even after 10 years. Great point.

FTZ you show in your post.

FTA:

functional form has units of pmol/L with inputs of TT and SHBG in nmol/L. Conversion to ng/dL as you show in your post.

I give FTZ the edge since you only need one equation. Good stuff.

 

[sammmy]

If what you say is correct, this graph would not exist:

View attachment 11234
View attachment 11236
View attachment 11237

Actually the first graph illustrates exactly the discrepancy between labs/methods (I assume both by dialysis). If the two labs were 'measuring the same thing' then y = x and the slope must have been 1. Instead, the slope is 1.32, which means 'Free T by API 4000' (the y value) is consistently 32% larger than the 'Referral lab' (the x value). The article cites 'correlation close to 1' which simply means there is a linear relationship between x and y, but not necessarily y = x.

That's why the values of free T that I get from Quest Diagnostics (uses Dialysis and Mass Spectrometry) had never been even close to the values from online calculators, including TruT, although they might be linearly related.

The point is, values of different labs are aiming at measuring the same free T but they succeed measuring just some proxy of it, not the absolute value of it.

 

[tareload]

Actually the first graph illustrates exactly the discrepancy between labs/methods (I assume both by dialysis). If the two labs were 'measuring the same thing' then y = x and the slope must have been 1. Instead, the slope is 1.32, which means 'Free T by API 4000' (the y value) is consistently 32% larger than the 'Referral lab' (the x value). The article cites 'correlation close to 1' which simply means there is a linear relationship between x and y, but not necessarily y = x.

That's why the values of free T that I get from Quest Diagnostics (uses Dialysis and Mass Spectrometry) had never been even close to the values from online calculators, including TruT, although they might be linearly related.

The point is, values of different labs are aiming at measuring the same free T but they succeed measuring just some proxy of it, not the absolute value of it.

Maybe this will help...

Observational error - Wikipedia

en.m.wikipedia.org

http://www2.ece.rochester.edu/courses/ECE111/error_uncertainty.pdf

There’s error involved in any measurement. Your conflating direct RIA free T methods which measure some quantity that may correlate with absolute free T (a proxy for free T) and dialysis free T method which attempts to measure actual free T.

Just because there is error involved in any measurement doesn’t mean the assay isn’t measuring the absolute quantity (or at least attempting to). There is no proxy variable involved with equilibrium dialysis coupled with LCMS/MS measurement of free T. There is a proxy variable involved with direct RIA free T measurement. All of this is academic but important to differentiate.

Also the best fit linear regression line in the plot above does not indicate that the API4000 measurement is consistently 32% higher that the reference lab. Clearly the Bland Altman bias plot shows this is far from the case. Instead what the ordinary least squares regression slope indicates is that the best fit line that minimizes the SSE (between the two method measurements in this study) has a slope of 1.32 (which assumes no error in the reference method). This can be affected by high leverage points / outliers, blah blah.

More info if you’d like:

9.1 - Distinction Between Outliers and High Leverage Observations | STAT 462

online.stat.psu.edu

Bland–Altman plot - Wikipedia

en.m.wikipedia.org

I showed yesterday in this thread that Vermeulen method was within approx. 20% of one of your dialysis free T results. Tru-T method way WAY overestimates (over 100%).

Respectfully, with your logic, when I take out a micrometer and measure the thickness of a sheet of paper I am not actually measuring the thickness of the sheet of paper but only a proxy. You are confusing proxy variable error with measurement error. If one day it is proven that free T does not exist (free hormone hypothesis would be disproven) and is not measurable, then I’ll buy your statement that dialysis + LCMS/MS is only a proxy for free T.

Make sense? We aren’t there yet, and our understanding of the physics of the situation indicates we can measure absolute free T (of course with the measurement error that comes along for the ride).

I now end my statistics lecture.

 

[madman]

Testosterone Deficiency in Men

Optimizing Diagnostic Accuracy and Treatment Decisions in Men With Testosterone Deficiency (2021) Shalender Bhasin, MB, BS *, Noelle Ozimek, MSc Abstract Objective: This narrative review offers a guideline-based approach for optimizing diagnostic evaluation and treatment decision-making in...

www.excelmale.com

 

[madman]

Harmonized reference ranges for Total and Free T levels

Accurate Measurement and Harmonized Reference Ranges for Total and Free Testosterone Levels (2022) Ravi Jasuja, Ph.D., Karol M. Pencina, Ph.D., Liming Peng, MSc, Shalender Bhasin, MB, BS* INTRODUCTION A central tenet of endocrinology is that the hormones secreted by the endocrine glands...

www.excelmale.com

 

[madman]

Improving Diagnostic Accuracy and Treatment Decisions in Men with Hypogonadism

www.excelmale.com

 

[madman]

CDC STANDARDIZED TOTAL T and ESTRADIOL ASSAYS and soon to be FREE TESTOSTERONE!

Measurement of Free Testosterone in Serum Using Equilibrium Dialysis Coupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices (2021) Hui Zhou, Ph.D., Ashley Ribera, BS, Amonae Dabbs-Brown, BS, Uliana Danilenko, Ph.D., Hubert W. Vesper, Ph.D. Centers for Disease Control and...

www.excelmale.com

* Limitations of using free testosterone by equilibrium dialysis and calculated free testosterone concentrations in practice are the lack of assay standardization, an accuracy-based quality control program, and a harmonized reference range. Until these limitations are addressed, free testosterone by equilibrium dialysis and calculated free testosterone should use reference ranges established by individual laboratories or their specific assay method




CDC Hormone Standardization Program (CDC HoSt) Certified Free Testosterone Procedures?

Sooner or later and SHBG to boot!

When the time comes these will be the only free testosterone assays I will recommend for men to use/rely upon when getting blood work done (pre/post-TRT).




*a lab/assay that is certified by the CDC's HoSt Program

The above applies to TT, estradiol, and soon enough free testosterone and SHBG!

 

[madman]

What to Measure: Testosterone or Free Testosterone?

What to Measure: Testosterone or Free Testosterone? (2021) Christina Wang and Ronald Swerdloff 1.1 Why Serum Testosterone Is Necessary for the Diagnosis of Testosterone Deficiency? Low serum testosterone (T) levels in adult men can lead to significant clinical symptoms and signs of...

www.excelmale.com

*The binding of T to SHBG is complex, which results in many different methods that directly measure or calculate free T. Some of these methods do not measure the free fraction of T and some formulae may provide less accurate results [40]

*Recent evidence suggests that the law of mass action formula which is based on the assumption that two T molecules bind to two binding sites on the SHBG with similar binding affinity may be incorrect. And further argues that the binding of T to SHBG may be a multistep, dynamic process with complex allosteric characteristics [65]. Based on this new model, investigators used a new formula to calculate free T in younger men in the Framingham Heart Study and showed that the newly calculated values were similar to those measured by equilibrium dialysis. They further verified that the calculated free T values had clinical diagnostic validity using data from the European Male Aging Study

*Currently, the CDC is developing a harmonized method for free T based on calculated free T using revised formulae. This may bring the measurement of free T to a referable standard in clinical laboratories and common reference intervals that all clinicians can use

*Perhaps the newer formula for calculated free T validated in multiple laboratories [65], will become generally available, correlate with free T by equilibrium dialysis and demonstrate improved correlation with clinical symptoms and therapeutic responsiveness. If all these prove to be true, then this formula to calculate free T may be a justified replacement for free T measurement by the equilibrium dialysis methodology



Phase II: Research and Commercialization of TruT Algorithm for Free Testosterone
Jasuja, Ravi

https://grantome.com/grant/NIH/R44-AG045011-02


The measurement of testosterone (T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women. Circulating T is bound with high affinity to sex hormone-binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active. Conditions that affect SHBG concentrations, such as aging and obesity, alter total but not free T concentrations; in these conditions, the determination of free T is necessary to obtain an accurate assessment of androgen status. The tracer analog method, the most widely used method for free T, has been shown to be inaccurate. The equilibrium dialysis method, considered the reference method, is technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that ?? the calculation of free testosterone is the most useful estimate of free testosterone in plasma?? Therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis. We have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterone's binding to SHBG using modern biophysical techniques. We have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer. Our computational framework incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T: SHBG association, and allostery

In phase I studies, we demonstrated that the TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogonadal men. We have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect, and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis. The phase I studies have led to the adoption of the TruTTM algorithm at several institutions.

The phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentrations, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (Aim 2). We will generate population-based reference ranges for free T (Aim 3). Phase II also includes plans for the commercialization of the TruTTM algorithm using a HIPAA-compliant infrastructure for its clinical adoption

The phase II program will provide validation of the TruTTM algorithm in the two most common clinical indications for free T measurement? men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders. It will also enable the development of a HIPAA-compliant platform that can be embedded into the electronic medical record for wider clinical adoption and for improving clinical care

 

[tareload]

Thanks for the update @madman. I can't wait to quit using my heuristic of 0-20% less than cfT-V to estimate fT via LC/MS-MS+ED:

Desperate advice needed quickly..too late?

First of all, I just want to thank all, for all the wealth of information that is shared here, more than anywhere else I have found on the internet. Excel Male, hands down the best! A special thanks to the moderators. Your knowledge on the subject of TRT, is better than any Dr. I have seen on...

www.excelmale.com

Does TRT reverse/improve sexual symptoms for most men with High Range SHBG / Lower Free T?

They're alluding to this study showing that low free testosterone is associated with the negative symptoms, even with normal total testosterone. I don't believe they are implying a causal connection to SHBG. Conclusions: Low cFT, even in the presence of normal TT, is associated with androgen...

www.excelmale.com

That would be really neat finding if major US reference laboratories are measuring fT wrong via equilibrium dialysis and the Tru-T approach is correct after all. As of now, it usually reads WAY high compared to anecdotal data on the internet for higher SHBG dudes.

Public Service Announcement: "Piss Poor" Direct RIA fT measurement (HELP is on the way)

Hey Folks, thought I would update the graph for free T reference ranges based on LC-MS/MS+ED and compare with Labcorp's direct RIA fT reference range. What the hell do you do with with your direct fT measurement if you want to compare with equibrium dialysis reference ranges? Take the Labcorp...

www.excelmale.com

 

[arseniol]

TrutT seems to give bad results with high SHBG, at least there are very far of what is obtained with RIA.

my case: Total 685 ng/ml, shbg:67 nmol/L

FTc(trut) -> 18.91 ng/dL
FTc(Vermellen/issam.ch) -> 9.29 ng/dL

FT(RIA) -> 11.0 pg/mL = 1100 pg/dL = 1.1 ng/dL

 

[Cataceous]

TrutT seems to give bad results with high SHBG, at least there are very far of what is obtained with RIA.

my case: Total 685 ng/ml, shbg:67 nmol/L

FTc(trut) -> 18.91 ng/dL
FTc(Vermellen/issam.ch) -> 9.29 ng/dL

FT(RIA) -> 11.0 pg/mL = 1100 pg/dL = 1.1 ng/dL

RIA is useless and should be ignored. Its reference range is also vastly different; you need to be looking at where values lie relative to their respective reference ranges. If you want a better comparison then you need to measure free testosterone by equilibrium dialysis. The two calculated methods are more or less in agreement, putting you at the low end of the healthy normal ranges: 16-31 ng/dL for Tru-T and maybe 10-20 ng/dL for Vermeulen.

 

[arseniol]

RIA is useless and should be ignored. Its reference range is also vastly different; you need to be looking at where values lie relative to their respective reference ranges. If you want a better comparison then you need to measure free testosterone by equilibrium dialysis. The two calculated methods are more or less in agreement, putting you at the low end of the healthy normal ranges: 16-31 ng/dL for Tru-T and maybe 10-20 ng/dL for Vermeulen.

Thanks so much, @Cataceous.

This high SHBG is pretty sad, it destroys a nice indigenous production.

Where may I find the ranges for Tru-T?

 

[FunkOdyssey]

This high SHBG is pretty sad, it destroys a nice indigenous production.

The idea of SHBG as a sponge that sucks up large amounts of testosterone and leaves your free T low doesn't seem to be how it really works. The way I understand it, high SHBG makes your meager production (revealed by the free T) look like it's normal or even high when it isn't. The SHBG is basically extending the half-life of the testosterone you've produced and the relationship seems to me alot like ferritin and iron.

 

[arseniol]

Thanks for your explanation, makes a lot of sense.

It would be interesting to have an index related to indigenous production. But due to this half-life point, it would be quite well following the free T value.

as far as I've seen Zakharov and TruT are estimating a lower impact of SHBG than Vermeulen.