Calculate free testosterone with TruT by FPT

readalot

Active Member
View attachment 7259
[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 to the new Multi-step Dynamic Binding Model with Complex Allostery developed in this study. These were utilized to obtain FT values (cFTZBJ) in samples obtained in clinical trials.






View attachment 7260
[0027] Figs. 8A-8B demonstrate that the binding of testosterone to SHBG displays complex allostery. Fig. 8A depicts a graph demonstrating that binding isotherms display significant non-linearity. Varying concentrations of testosterone were incubated with a fixed concentration of SHBG (5, 10 or 20 nM) and bound testosterone was plotted against total testosterone concentration. The binding isotherms were generated at 5, 10 and 20 nM SHBG. Curves represent the result of the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery.




View attachment 7261
Fig. 8B depicts a graph demonstrating that depletion of FT by varying SHBG concentration is best described by the new Multi-step Dynamic Binding Model with Complex Allostery. Constant concentration of testosterone (6, 12 , 17 or 32 nM) was incubated with increasing SHBG concentrations, and free testosterone concentration in buffer side was plotted against SHBG concentration. The curves are the result of the fit of data to the new Multi-step Dynamic Binding Model with Complex Allostery.




View attachment 7262
[0028] Figs. 9A-9C depicts graphs depicting the fits of the various models of testosterone's binding to SHBG to the experimental data from binding isotherms, depletion experiments, and ITC. Left panels: The figures show the fits of data to the various models examined in this study.Right panels: The figures show corresponding residuals of the fit of data to various models of testosterone's binding to SHBG. Neither the Vermeulen's equation nor the simple allostery models adequately fit the experimental data from binding isotherms, depletion experiments, or ITC. The new Multi-step Dynamic Binding Model with Complex Allostery (model E) provided the optimal fit to the experimental data from all three methods.




View attachment 7263
[0029] Fig. 10 depicts a schematic of the control of testosterone levels.




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[0030] Fig. 11 depicts a schematic of an exemplary system of determining free testosterone levels and/or dosages.




View attachment 7265
[0031] Fig. 12 depicts a device or a computer system 1000 comprising one or more processors 1300 and a memory 1500 storing one or more programs 1600 for execution by the one or more processors 1300.
It would be an interesting plot to pool all the published data over last 10 years for equilibrium dialysis data points and then also plot Vermeulen and Tru-T on same plot. You could plot against total T or SHBG. Unless I am missing something you are going to get a cluster of data points that are very close to Vermeulen and a country mile away from Tru-T. Then another cluster that sit on top of Tru-T line. What does that say about the agreement between the various equilibrium dialysis measurement data between labs in the papers?

I conclude that if you had access to all those blood samples and ran head to head equilibrium dialysis tests you will get very different numbers. My apologies if I missed this discussion some where else. Something ain’t right with the interlab agreement on the equilibrium dialysis data.
 

readalot

Active Member
It would be an interesting plot to pool all the published data over last 10 years for equilibrium dialysis data points and then also plot Vermeulen and Tru-T on same plot. You could plot against total T or SHBG. Unless I am missing something you are going to get a cluster of data points that are very close to Vermeulen and a country mile away from Tru-T. Then another cluster that sit on top of Tru-T line. What does that say about the agreement between the various equilibrium dialysis measurement data between labs in the papers?

I conclude that if you had access to all those blood samples and ran head to head equilibrium dialysis tests you will get very different numbers. My apologies if I missed this discussion some where else. Something ain’t right with the interlab agreement on the equilibrium dialysis data.
Here we go:


Zakharov (2015)


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Frozen samples never thawed:
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Fiers 2018:

Assay procedures

Assay of serum SHBG levels was performed on an E170 Modular immunoassay analyzer and of albumin was performed on the Cobas 8000 (Roche Diagnostics; Indianapolis, IN). Assay of total T in serum and direct measurement of FT in dialysate after ED against serum as described later in Methods, was done by LC-MS/MS on an AB Sciex 5500 triple-quadrupole mass spectrometer (Toronto, ON, Canada), as previously described (22). This method was validated for T against an isotope dilution mass spectrometry candidate reference method (23) using a common serum panel, and it also has recently been compared with a reference method at the Centers for Disease Control and Prevention (Atlanta, GA) (24).

Serum FT level was determined by a direct dialysis method (i.e., ED of undiluted serum against buffer with direct LC-MS/MS assay of FT in the dialysate). ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA). Serum (men, 500 μL; women, 1000 μL) was dialyzed at 37°C for 24 hours at pH 7.28 using protein-free buffer prepared according to Yue et al. (25).

For assay of total T, a liquid–liquid extraction was performed on 100 µL serum samples. Interassay coefficient of variation for T is 6.5% at 3 ng/dL (104 pmol/L; n = 30) with a limit of quantitation of 1 ng/dL (35 pmol/L). For measurement of FT by direct ED, interassay coefficient of variability was 13.5% at 0.18 ng/dL (6.2 pmol/L) with a limit of quantitation of 0.07 ng/dL (2.4 pmol/L). Experiments on various buffer and serum volumes, nonspecific binding, and mass spectrometry ion suppression all yielded excellent target recoveries within 5% tolerance. Details are provided in a validation summary in the Supplemental Validation Summary.


Check out the Validation summary:






1603342765919.png



Now superimpose the % free T data from these two papers and plot vs SHBG or TT. What am I missing? Who didn't properly validate their free T ED assay? If you sent a sample and ran it on these two machines you would get drastically different free T by "gold standard" ED method.
 
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readalot

Active Member
In fact I do expect a little rise post-injection in these cases, but it's not enough to affect the results. I did measure consistent values pre-injection and day-after injection on the EOD schedule.


The literature on TRT does not support the notion of arbitrary variations in T or free T. The creators of free T calculations believe that it's possible to obtain a fairly accurate value for free testosterone based on total testosterone, SHBG, etc. If you prefer to reject that then it's beyond my purview. But the calculation of free T is not essential to the underlying principles; it just allows a nice demonstration of them. The Law of Mass Action is accepted theory, and harder to reject.

There are two basic principles: 1) If we're putting testosterone in at a steady rate then at steady state it's being used up at the same rate. 2) The rate of use is proportional to the free hormone concentration. Therefore, free testosterone is proportional to the amount of testosterone we're putting in.

Is there additional complexity beyond this model? Most likely. Does it matter in most cases? Probably not.


This is par for the course. Different assays produce very different results. The direct assays are typically giving results around 10-30 pg/mL, which is off by a factor of 10 from the true concentrations. It would be ok if they correlated well with more accurate tests, but they don't do that either. The point of the Tru-T calculator is attain better correlation with the most accurate testing method, which it does.
Maybe you've seen this (Sept 2020). Interesting discussion:
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Discussion
The findings of this study provide further support for the idea that the mechanism for the strong positive correlation between the circulating levels of SHBG and total testosterone in men is more complex than the stoichiometry of a high affinity circulating transport protein which controls metabolic clearance, and its ligand. Instead, the positive correlation in untreated, but not in testosterone-treated men, implies that SHBG regulates testicular negative feedback either directly or by modulating the entry of testosterone or estradiol into cells in the hypothalamus and/or pituitary to control gonadotropin synthesis and secretion, and thereby testosterone levels.
 

readalot

Active Member
Maybe you've seen this (Sept 2020). Interesting discussion:
View attachment 11202

Discussion
The findings of this study provide further support for the idea that the mechanism for the strong positive correlation between the circulating levels of SHBG and total testosterone in men is more complex than the stoichiometry of a high affinity circulating transport protein which controls metabolic clearance, and its ligand. Instead, the positive correlation in untreated, but not in testosterone-treated men, implies that SHBG regulates testicular negative feedback either directly or by modulating the entry of testosterone or estradiol into cells in the hypothalamus and/or pituitary to control gonadotropin synthesis and secretion, and thereby testosterone levels.

Methods
Medical records were reviewed retrospectively from adult men who presented for a clinical evaluation of adult onset hypogonadism (n = 33), and from adult men who were being treated for hypogonadism with transdermal testosterone (n = 25).
The men with AOH presented for evaluation because of erectile dysfunction, low libido and/or asthenia. A thorough medical history and physical examination were performed, and classical causes of hypogonadism were excluded, in most cases by additional endocrine testing. Testis size was ≥ 20 mL. 6 men had diabetes, and 16 were being treated for dyslipidemia.
Testosterone-treated men had been diagnosed with adult onset hypogonadism (n = 12), hypopituitarism (n = 9), congenital hypogonadotropic hypogonadism (n = 2), or Klinefelter syndrome (n = 2), and were being treated with a stable dose of a transdermal testosterone preparation.
Assays were performed at Quest Diagnostics. Testosterone levels were measured by liquid chromatography-mass spectrometry, and SHBG was measured by immunoassay. Free testosterone was measured by indirect equilibrium dialysis. Laboratory reference ranges for adult men are: 250–1100 ng/dL for total testosterone, 10–50 nmol/L for SHBG, and 35–155 pg/mL for free testosterone.
Statistical analysis
Two group comparisons were performed using Student’s t-test or the Mann–Whitney Rank Sum test when data were not normally distributed or groups had unequal variance. Pearson correlation coefficients were calculated using SigmaStat/Systat Software, Inc. (San Jose, CA). Data are presented as the mean ± SD or the median and 25–75% range.







Try fitting these data with either Vermeulen or Tru-T:
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The only ED free T data I can find in the literature that fits the Tru-T calculator is the data presented in the Tru-T papers. All the other free T ED data I've scanned (again not exhaustive) shows Vermeulen overpredicts and Tru-T WAY overpredicts measured free T.

You guys have other parity plots vs Tru-T vs Vermeulen with equilibrium dialysis data?
 
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readalot

Active Member
Now does everyone truly know where their free testosterone levels sit and how many are experiencing negative effects/sides on said protocol only to end up chasing their tale from unknowingly having too high FT levels.






Assessment of free testosterone concentration (2019)

Brian G Keevil, Jo Adaway





*The older, direct analogue RIA methods have been discredited and are no longer recommended for use (6-8).
Great point on the direct free T RIA measurement. From the same paper:

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1603378712105.png


We still have either an accuracy or bias issue in the measurement of free T by equilibrium dialysis. The vast majority of data presented in the literature indicates both linear Vermeulen and Tru-T way overestimate free T measured by equilbrium dialysis. Vermeulen overestimates less.

Whether the free hormone hypothesis is correct or not, my takeaway is there's a serious issue with measuring free testosterone between labs and assays (all employing "gold standard" ED). Before you validate the fitness of a model, you have to validate the accuracy of the data.
 

readalot

Active Member
My latest measurement, not using any androgens:
Total Testosterone 711 ng/dL
SHBG 58 nmol/L
Albumin 4.8 g/L

TruT Free Testosterone calculated: 19.84ng/dL = 198.4 pg/mL, with unspecified normal range?

Free Testosterone (Dialysis test by Quest Diagnostics): 78.2pg/mL with normal range 35-155.

The obvious issue is that if TruT has the same normal range, it turns out I have free testosterone above the normal range.
On the other hand, I have delayed ejaculation, not being able to put a lot of muscle by exercising, some flat around the navel, which contradicts any claim for "high free testosterone".
Free T calculated by Vermeulen with same inputs:
1603379150992.png


Again, same picture as the papers I have shared.
 

readalot

Active Member
Same thing here. Vermeulen vs dialysis data above in a parity plot would show much closer agreement.
Another example:


FT by "dialysis" 6.5 ng/dL
Vermeulen 11.8 ng/dL
Tru-T 27 ng/dL

Anyone have Labcorp free T data with ED (along with TT by LCMS and SHBG)?
 

readalot

Active Member
Another example:


FT by "dialysis" 6.5 ng/dL
Vermeulen 11.8 ng/dL
Tru-T 27 ng/dL

Anyone have Labcorp free T data with ED (along with TT by LCMS and SHBG)?
Another example. Seriously, a grad student could scour the web and put together a retrospective ad hoc analysis:


SHBG 83
testosterone free (dialysis) and total, MS
testosterone total, MS 508 range 250-1100 nq/dl
testosterone free 47.2 range 35-155 pq/ml

Vermeulen: 5.47 ng/dL
Tru-T: 12.25 ng/dL
 

Cataceous

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

readalot

Active Member

Another example. Seriously, a grad student could scour the web and put together a retrospective ad hoc analysis:


SHBG 83
testosterone free (dialysis) and total, MS
testosterone total, MS 508 range 250-1100 nq/dl
testosterone free 47.2 range 35-155 pq/ml

Vermeulen: 5.47 ng/dL
Tru-T: 12.25 ng/dL

More data (intermixed with direct fT data which will confuse most).
 

readalot

Active Member

More to read for the truly touched researcher.

Huge gap between equilibrium dialysis free T measurement (for many guys) and Tru-T (Vermeulen still overestimates 20-30%).

@Nelson Vergel, we should design a DOE and send member blood samples to Quest and Labcorp and have them run equilibrium dialysis / LCMS and do a parity plot. We wouldn't have a reference standard but it may prove the point. Guys really don't know what their free T value is.
 
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readalot

Active Member
It does seem like a significant discrepancy. Maybe we should just revert to using one of the empirical formulas mentioned in your other post:
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?
That's where I'm headed till we figure out where this discrepancy is coming from:



Endocrine Sciences reference range runs from 5.2 to 28 ng/dL (now offered through Labcorp):

1603381549925.png
 

readalot

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

1603382756053.png

Excerpt:

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1603382836413.png


Seems like since they charge ~$106 a pop for LCMS TT and ED Free T, Labcorp and Quest could both publish a reference standard calibration plot (harmonization plot) to show their customers what they are getting and what they are paying for.
 

readalot

Active Member
To your point:

View attachment 11213
Excerpt:

View attachment 11212

View attachment 11214View attachment 11215

Seems like since they charge ~$106 a pop for LCMS TT and ED Free T, Labcorp and Quest could both publish a reference standard calibration plot (harmonization plot) to show their customers what they are getting and what they are paying for.
Mayo Reference Laboratory Range for free Testosterone by ED:


Method Description

Total Testosterone:

Deuterated stable isotope (d3-testosterone) is added to a 0.2-mL serum sample as an internal standard. Protein is precipitated from the mixture by the addition of acetonitrile. The testosterone and internal standard are extracted from the resulting supernatant by an online extraction utilizing high-throughput liquid chromatography (HTLC). This is followed by conventional liquid chromatography and analysis on a tandem mass spectrometer equipped with a heated nebulizer ion source.(Wang C, Catlin DH, Demers LM, et al: Measurement of total testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2004;89:534-543; Taieb J, Mathian B, Millot F, et al: Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003;49:1381-1395)



Free Testosterone:

This method is based on equilibrium dialysis in which free-labeled testosterone is allowed to pass through a semipermeable membrane, whereas testosterone bound to the sex hormone-binding globulin (SHBG) remains inside the dialysis tubing. After dialysis, radioactivity is measured both inside and outside the tubing; the free testosterone results are expressed as a percentage of total testosterone. The resulting percentage is multiplied by the total testosterone concentration obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and an absolute free testosterone value is obtained.(Vermeulen A, Stoica T, Verdonck L: The apparent free testosterone concentration, an index of androgenicity. J Clin Endocrinol Metab. 1971;33:759-767; Bammann BL, Coulam C, Jiang NS: Total and free testosterone during pregnancy. Am J Obstet Gynecol. 1980;137:293-298)



Bioavailable Testosterone:

The method is based on the differential precipitation of SHBG by ammonium sulfate following equilibration of the serum specimen and tracer amounts of tritium-labeled testosterone. The results are expressed as the percent of testosterone free or albumin bound (not precipitated with SHBG) compared to an albumin standard. The product of this percentage and the total testosterone measurement is the total bioavailable testosterone.(Wheeler MJ: The determination of bioavailable testosterone. Ann Clin Biochem. 1995;32:345-357)


Reference Values

TESTOSTERONE, TOTAL

Males

0-5 months: 75-400 ng/dL

6 months-9 years: <7-20 ng/dL

10-11 years: <7-130 ng/dL

12-13 years: <7-800 ng/dL

14 years: <7-1,200 ng/dL

15-16 years: 100-1,200 ng/dL

17-18 years: 300-1,200 ng/dL

> or =19 years: 240-950 ng/Dl

Tanner Stages*

I (prepubertal): <7-20

II: 8-66

III: 26-800

IV: 85-1,200

V (young adult): 300-950

TESTOSTERONE, FREE


Males (adult):

20-<25 years: 5.25-20.7 ng/dL

25-<30 years: 5.05-19.8 ng/dL

30-<35 years: 4.85-19.0 ng/dL

35-<40 years: 4.65-18.1 ng/dL

40-<45 years: 4.46-17.1 ng/dL

45-<50 years: 4.26-16.4 ng/dL

50-<55 years: 4.06-15.6 ng/dL

55-<60 years: 3.87-14.7 ng/dL

60-<65 years: 3.67-13.9 ng/dL

65-<70 years: 3.47-13.0 ng/dL

70-<75 years: 3.28-12.2 ng/dL

75-<80 years: 3.08-11.3 ng/dL

80-<85 years: 2.88-10.5 ng/dL

85-<90 years: 2.69-9.61 ng/dL

90-<95 years: 2.49-8.76 ng/dL

95-100+ years: 2.29-7.91 ng/dL
 

readalot

Active Member
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:

1603384846658.png



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
 

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readalot

Active Member

Prevailing calculators are inaccurate

It has been demonstrated that the calculated free testosterone derived using commonly available online calculators, which are based on simpler models assuming simple 1:1 binding of testosterone and SHBG [1,2], deviate from measurements obtained by equilibrium dialysis by as much as 40% [3-9].



This is a true statement. The only issue is that Tru-T is even more inaccurate than the simple linear models.


The purported reference range for Adult male free T (16-31 ng/dL) based on Tru-T model does not match the reference ranges from 4 major reference laboratories in the US.


Compare the ranges in the paper below to the reference ranges from US reference laboratories I just shared above. Was there an artifact in the equilibrium dialysis method the authors used to validate their model? The ranges below are higher (and low is skewed higher) compared to US reference laboratories.

Gen 3 reference range below converted to nd/dL: 16.3-31.3



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