Reference Intervals for Free Testosterone in Adult Men Measured Using a Standardized Equilibrium Dialysis Procedure

[Cataceous]

...
I am too impatient today to wait for the next paper so here you go. You got it here first on @Nelson Vergel 's ExcelMale.

Given the Tru-T website locks you down after a few tries I didn't fill out the whole table but covered the boundaries:
...

Very nice work! Shows why cfTV sometimes makes more sense than cfTZ.

By the way, the Tru-T lockdown is trivially defeated by deleting the site's cookies and reloading the page after each expiration. In Safari, for example, in Preferences->Privacy->Manage Website Data...:

 

[tareload]

Very nice work! Shows why cfTV sometimes makes more sense than cfTZ.

By the way, the Tru-T lockdown is trivially defeated by deleting the site's cookies and reloading the page after each expiration. In Safari, for example, in Preferences->Privacy->Manage Website Data...:
View attachment 26319

Thank you! May use that to complete the table. At some point based on eyeballing the residuals I would imagine you could adjust the constant(s) and get a very good residual fit with a corrected cfTV model. Down the road after the standardization is complete. Qualitatively the fit is quite good vs these new data. Lots of papers to be published on all this I would imagine. Very exciting.

There is hope guys will be able to come out of the darkness and avoid the perpetual darkness of free T world.

 

[tareload]

Poor man's superposition of Fiers 2018 data set vs this latest one:

Comparing yellow data points (Fiers 2018) to new data you can see huge discrepancy as I have wondered about. See Figs. 1A and 1B in the original post. You can see a huge multi percent discrepancy for measured fT across the range of SHBG.

@sammmy this is what I was getting at in the other thread a while back.

 

[tareload]

Hmmm, this doesn't explain the discrepancy. Now we are deep water.

Fiers 2018:

Jasuja 2022:

Now we need the paper comparing Fiers 2018 vs Jasuja 2022.

 

[tareload]

Again for completeness, the graphs I did above vs Fiers 2018 data.


cfTV:

Tru-T:

Will the true fT measurement please stand up?

Poor dudes. This just ain't right for the regular Joe trying to do his best.

 

[FunkOdyssey]

Tru-T:
View attachment 26325

I'm no data scientist but Tru-T looks like hot garbage on this graph.

 

[tareload]

At some point based on eyeballing the residuals I would imagine you could adjust the constant(s) and get a very good residual fit with a corrected cfTV model. Down the road after the standardization is complete. Qualitatively the fit is quite good vs these new data. Lots of papers to be published on all this I would imagine. Very exciting.

@Cataceous

Following up on comment above...a 1.9 multiplier on cfTV easily collapses all the new data into the model envelope:

Now we are back to scratching our heads which is the true fT via ED data? See above for comparisons of latest paper vs Fiers 2018. Both papers report incubation at 37 deg C for 24 hrs.

 

[tareload]

I'm no data scientist but Tru-T looks like hot garbage on this graph.

This one is the critical one:


Compare yellow data (2018) vs new blue, red, green data (2022). I would have to really get into the weeds to understand the huge difference as both assays report 37 deg C /24 hr incubation. Will have to look at that when I have more time. This is a black hole haha.

If I was reviewing this paper I surely would have asked to compare data generated vs Fiers 2018 data set and explain to the reader the disrepancy (or at least attempt to). This seems reasonable given the paper's title...

Reference Intervals for Free Testosterone in Adult Men Measured Using a Standardized Equilibrium Dialysis Procedure.​

This purported quotation seems applicable.

 

[Cataceous]

...
Compare yellow data (2018) vs new blue, red, green data (2022). I would have to really get into the weeds to understand the huge difference as both assays report 37 deg C /24 hr incubation. Will have to look at that when I have more time. This is a black hole haha.

If I was reviewing this paper I surely would have asked to compare data generated vs Fiers 2018 data set and explain to the reader the disrepancy (or at least attempt to). This seems reasonable given the paper's title...

With respect to practical applications it does appear that absolute FT numbers remain elusive and somebody has some explaining to do. At least cfTV still looks good for relative agreement, so that's what I'll continue to rely on as a quasi-standard.

 

[tareload]

With respect to practical applications it does appear that absolute FT numbers remain elusive and somebody has some explaining to do. At least cfTV still looks good for relative agreement, so that's what I'll continue to rely on as a quasi-standard.

To be honest, disappointed some internet moron (me) outside this field has to put together a poor man's plot of these data to show we still have no idea what the real absolute fT numbers are. In my field this new paper would be considered half baked and discussion incomplete without comparison to existing literature of which Fiers 2018 paper is critical prior work. What have we learned since 2018 given the collaboration of the various authors and their respective institutions? Come on PIs, talk to each other!

@madman, can you please provide the reference list for the new paper? I'd like to read the discussion and methods sections again with the ref list in hand.

Sorry guys, back to griping about Labcorp vs Quest ED vs what the hell is direct fT measurement. I tried.

 

[tareload]

Corresponding authors on the two papers if someone would like to notify the respective corresponding authors and ask them to take a look at this thread and comment. Nelson (@Nelson Vergel) if you would like to do the honors I would (and I am sure many others) would be grateful.


@bennettjc I know you are always game. Nothing like a little peer review on one's scholarship from ExcelMale.

Anyone else? I prefer to stay anonymous.

===
Fiers 2018:

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

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

Tom Fiers
Department of Clinical Chemistry, Ghent University Hospital, Ghent, Belgium

Correspondence and Reprint Requests: Tom Fiers, MD, University Hospital Ghent, 2P8, De Pintelaan185, 9000 Ghent, Belgium. E-mail: [email protected].

===
Jasuja 2022:

https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310

Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure​

Shalender Bhasin
[email protected]
Research Program in Men's Health: Aging and Metabolism; Boston Claude D. Pepper Older Americans Independence Center; Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

Correspondence

Shalender Bhasin, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, BLI-541, Boston, MA 02115, USA.

Email: [email protected]

===

 

[madman]

To be honest, disappointed some internet moron (me) outside this field has to put together a poor man's plot of these data to show we still have no idea what the real absolute fT numbers are. In my field this new paper would be considered half baked and discussion incomplete without comparison to existing literature of which Fiers 2018 paper is critical prior work. What have we learned since 2018 given the collaboration of the various authors and their respective institutions? Come on PIs, talk to each other!

@madman, can you please provide the reference list for the new paper? I'd like to read the discussion and methods sections again with the ref list in hand.

Sorry guys, back to griping about Labcorp vs Quest ED vs what the hell is direct fT measurement. I tried.

Everyone jumping to conclusions already yet as I have stated numerous times there is much more to come.

Going to be a while before the s**t show comes to an end!

Fiers 2018/Jasuja 2022 paper.

Although both direct ED methods for testing FT were performed under standardized conditions the ED devices used were different.

Would not get too caught up on just the temperature/incubation times as there is much more going on.

Remember buffer composition, incubation time, and temperature will have a big impact on the free testosterone concentrations.

Throw in other assay conditions too!

Not sure what buffer composition was used in Fiers study.

As you can see the membrane MWCO (KDa) was different let alone the type of system.


ED Device Fiers 2018
Description Harvard Apparatus Micro-Equillibrium Dialyzer System, 1 mL cells
Maximum Serum Capacity (mL) 1 mL
Ratio of sample/buffer volume ?
Membrane Composition RC
Membrane MWCO (KDa) 25
Incubation Time (hr) 24 hrs
Type of System Individual unit, assembly required
Automation compatible No



ED Device Jasuja 2022
Description Harvard Apparatus DispoEquillibrium Dialyzers
Maximum Serum Capacity (mL) 0.2 mL
Ratio of sample/buffer volume 1:1
Membrane Composition RC
Membrane MWCO (KDa) 10
Incubation Time (hr) 24 hrs
Type of System 96-well built-in plate
Automation compatible Difficult




Jasuja 2022 (https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310)

*Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (41,43)


41. Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.

43. Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414




This is key!

*Our normative ranges are similar to those reported by pioneers of this field using the legacy method (2) but the percent free testosterone in our reference sample differs from that reported by another laboratory (10,52); this difference in percent free testosterone from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods (10,52). The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time (53,54); because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.


10. Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab. 1996;81(5):1821-1826.

52. Fiers T, Wu F, Moghetti P, Vanderschueren D, Lapauw B, Kaufman JM. Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis. J Clin Endocrinol Metab. 2018;103(6):2167- 2174




Fiers 2018

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 different cal

academic.oup.com

*ED was performed using Fast Micro-Equilibrium dialyzer cartridges and regenerated cellulose 25 kDa membranes (Harvard Apparatus, Holliston, MA).








Jasuja 2022 paper:

In an effort to generate reference intervals for free testosterone concentrations in men that can be used across laboratories, we report here a detailed description of a non-proprietary, standardized equilibrium dialysis procedure that can be replicated in any qualified laboratory using commercially available reagents. Instead of using a tritium-labeled tracer, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to directly measure testosterone concentrations on both sides of the dialysis chamber. We employed standardized dialysis conditions at 37ºC for a duration of time shown to be sufficient for reaching equilibrium. Using this validated equilibrium dialysis method, we evaluated the distribution of free testosterone concentrations in a prospectively-collected sample of community-living healthy nonobese men, 19 years or older. The blood samples were collected in the morning before 10 AM after an overnight fast. Total testosterone levels in the serum as well as in the dialysate were measured using a validated LC-MS/MS assay that is certified by the Hormone Standardization Program for Testosterone (HoST) of the US Centers for Disease Control and Prevention (42). We report the distribution of free testosterone concentrations in healthy young men, 19 to 39 years, as well by age groups, generated using this standardized equilibrium dialysis procedure.

Free testosterone concentrations in human serum samples were determined using a standardized protocol of equilibrium dialysis coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for testosterone. Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (41,43). The dialysis buffer composition simulated the ionic strength conditions of the human plasma: 90 mM NaCl, 3 nM KCl, 1.3 mM KH2PO4, 1.9 mM, 1.1 mM MgSO4·7H2O, 5 mM 0.30 g urea and 23 mM HEPES sodium salt, 30 nM HEPES acid, 8 mM sodium azide, and 1 mL of 0.06% DL lactic acid. 200 µL of dialysis buffer was added to the “buffer side” and 200 µL of undiluted serum was added to the “sample side”.


41. Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.

43. Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414.




To standardize the dialysis procedure, in preliminary experiments, we determined the time required to achieve equilibrium in the dialysis and compared the effect of incubation at room temperature versus 37ºC. After the dialysis conditions had been standardized in these preliminary experiments, the dialysis plates were incubated for 24 hours at 37ºC, after which 150 µL aliquots were removed from each side for testosterone measurement using a validated LC-MS/MS assay that is certified by the Center for Disease Control’s Hormone Standardization Program (HoST) (44,45). The dialysis plates were rotated throughout the course of the experiment to disrupt the formation of the Nernst diffusion layer and minimize its influence on the diffusion rate.


44. Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, Gill TM, Barrett-Connor E, Swerdloff RS, Wang C, Ensrud KE, Lewis CE, Farrar JT, Cella D, Rosen RC, Pahor M, Crandall JP, Molitch ME, Cifelli D, Dougar D, Fluharty L, Resnick SM, Storer TW, Anton S, Basaria S, Diem SJ, Hou X, Mohler ER, 3rd, Parsons JK, Wenger NK, Zeldow B, Landis JR, Ellenberg SS, Testosterone Trials I. Effects of Testosterone Treatment in Older Men. N Engl J Med. 2016;374(7):611-624.

45. Travison TG, Vesper HW, Orwoll E, Wu F, Kaufman JM, Wang Y, Lapauw B, Fiers T, Matsumoto AM, Bhasin S. Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe. J Clin Endocrinol Metab. 2017;102(4):1161-1173.







Again when it comes to the confusion of the currently used/relied upon ED methods between laboratories for testing free testosterone everyone needs to keep in mind that not all ED devices/procedures are the same.

Sums up the s**tshow!


Jasuja paper:

*An expert panel of the Endocrine Society (9) reviewed the various methods for determining free testosterone (4,5,9,21-32) and concluded that each method has some inherent limitations but that the equilibrium dialysis method is the reference standard against which all other methods should be compared (33). However, substantial heterogeneity in the procedures used by various laboratories for performing the equilibrium dialysis assay has contributed to variability in the reported free testosterone values

*Furthermore, the determination of free testosterone concentration by equilibrium dialysis is affected greatly by the assay conditions, including the buffer composition, the incubation time, and the temperature

*The accuracy and precision of the total testosterone assay also affect the precision and accuracy of the measured free testosterone concentration. Most commercial laboratories do not report the buffer composition and other dialysis conditions which renders it difficult to evaluate their methods; the procedures for equilibrium dialysis have varied even in published reports from academic research laboratories (2,24,32-37). Because of the wide variation in the equilibrium dialysis procedures, the reference ranges are not generalizable across laboratories


22. Morley JE, Patrick P, Perry HM, 3rd. Evaluation of assays available to measure free testosterone. Metabolism: clinical and experimental. 2002;51(5):554-559.

24. Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J Clin Endocrinol Metab. 1998;83(4):1312-1318.

32. Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982;16(6):801-810.

33. Ly LP, Handelsman DJ. Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. Eur J Endocrinol. 2005;152(3):471-478.

34. Hammond GL, Nisker JA, Jones LA, Siiteri PK. Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis. J Biol Chem. 1980;255(11):5023-5026.

35. Pirke KM, Doerr P. Age related changes in free plasma testosterone, dihydrotestosterone and oestradiol. Acta Endocrinol (Copenh). 1975;80(1):171-178.

36. Umstot ES, Baxter JE, Andersen RN. A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. J Steroid Biochem. 1985;22(5):639-648.

37. Swerdloff RS, Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Longstreth J, Berman N. Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab. 2000;85(12):4500-4510.

 

[madman]

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11. Bhasin S, Brito JP, Cunningham GR, Hayes FJ, Hodis HN, Matsumoto AM, Snyder PJ, Swerdloff RS, Wu FC, Yialamas MA. Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018;103(5):1715-1744.

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23. Rinaldi S, Geay A, Dechaud H, Biessy C, Zeleniuch-Jacquotte A, Akhmedkhanov A, Shore RE, Riboli E, Toniolo P, Kaaks R. Validity of free testosterone and free estradiol determinations in serum samples from postmenopausal women by theoretical calculations. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2002;11(10 Pt 1):1065-1071.

24. Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women. J Clin Endocrinol Metab. 1998;83(4):1312-1318.

25. Van Uytfanghe K, Stockl D, Kaufman JM, Fiers T, Ross HA, De Leenheer AP, Thienpont LM. Evaluation of a candidate reference measurement procedure for serum-free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry. Clin Chem. 2004;50(11):2101-2110.

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28. Mazer NA. A novel spreadsheet method for calculating the free serum concentrations of testosterone, dihydrotestosterone, estradiol, estrone, and cortisol: with illustrative examples from male and female populations. Steroids. 2009;74(6):512-519.

29. Miller KK, Rosner W, Lee H, Hier J, Sesmilo G, Schoenfeld D, Neubauer G, Klibanski A. Measurement of free testosterone in normal women and women with androgen deficiency: comparison of methods. The Journal of clinical endocrinology and metabolism. 2004;89(2):525-533.

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31. Ramlau-Hansen CH, Hansen M, Jensen CR, Olsen J, Bonde JP, Thulstrup AM. Semen quality and reproductive hormones according to birthweight and body mass index in childhood and adult life: two decades of follow-up. Fertility and sterility. 2010;94(2):610-618.

32. Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982;16(6):801-810.

33. Ly LP, Handelsman DJ. Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. Eur J Endocrinol. 2005;152(3):471-478.

34. Hammond GL, Nisker JA, Jones LA, Siiteri PK. Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis. J Biol Chem. 1980;255(11):5023-5026.

35. Pirke KM, Doerr P. Age-related changes in free plasma testosterone, dihydrotestosterone and oestradiol. Acta Endocrinol (Copenh). 1975;80(1):171-178.

36. Umstot ES, Baxter JE, Andersen RN. A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. J Steroid Biochem. 1985;22(5):639-648.

37. Swerdloff RS, Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Longstreth J, Berman N. Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. J Clin Endocrinol Metab. 2000;85(12):4500-4510.

38. Wu FC, Tajar A, Beynon JM, Pye SR, Silman AJ, Finn JD, O'Neill TW, Bartfai G, Casanueva FF, Forti G, Giwercman A, Han TS, Kula K, Lean ME, Pendleton N, Punab M, Boonen S, Vanderschueren D, Labrie F, Huhtaniemi IT, Group E. Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med. 2010;363(2):123-135.

39. Mellstrom D, Johnell O, Ljunggren O, Eriksson AL, Lorentzon M, Mallmin H, Holmberg A, Redlund-Johnell I, Orwoll E, Ohlsson C. Free testosterone is an independent predictor of BMD and prevalent fractures in elderly men: MrOS Sweden. J Bone Miner Res. 2006;21(4):529-535.

40. Yeap BB, Marriott RJ, Antonio L, Chan YX, Raj S, Dwivedi G, Reid CM, Anawalt BD, Bhasin S, Dobs AS, Hankey GJ, Matsumoto AM, Norman PE, O'Neill TW, Ohlsson C, Orwoll ES, Vanderschueren D, Wittert GA, Wu FCW, Murray K. Serum Testosterone is Inversely and Sex Hormone-binding Globulin is Directly Associated with All-cause Mortality in Men. J Clin Endocrinol Metab. 2021;106(2):e625-e637.

41. Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.

42. Cao ZT, Botelho JC, Rej R, Vesper H. Accuracy-based proficiency testing for testosterone measurements with immunoassays and liquid chromatography-mass spectrometry. Clin Chim Acta. 2017;469:31-36.

43. Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414.

44. Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, Gill TM, Barrett-Connor E, Swerdloff RS, Wang C, Ensrud KE, Lewis CE, Farrar JT, Cella D, Rosen RC, Pahor M, Crandall JP, Molitch ME, Cifelli D, Dougar D, Fluharty L, Resnick SM, Storer TW, Anton S, Basaria S, Diem SJ, Hou X, Mohler ER, 3rd, Parsons JK, Wenger NK, Zeldow B, Landis JR, Ellenberg SS, Testosterone Trials I. Effects of Testosterone Treatment in Older Men. N Engl J Med. 2016;374(7):611-624.

45. Travison TG, Vesper HW, Orwoll E, Wu F, Kaufman JM, Wang Y, Lapauw B, Fiers T, Matsumoto AM, Bhasin S. Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe. J Clin Endocrinol Metab. 2017;102(4):1161-1173.

46. Elveback L. The population of healthy persons as a source of reference information. Hum Pathol. 1973;4(1):9-16.

47. Solberg H. Approved recommendations (1987) on the theory of reference values, II: selection of individuals for the production of reference values. . Clin Chem Acta. 1987;25:639-644.

48. Bhasin S, Pencina M, Jasuja GK, Travison TG, Coviello A, Orwoll E, Wang PY, Nielson C, Wu F, Tajar A, Labrie F, Vesper H, Zhang A, Ulloor J, Singh R, D'Agostino R, Vasan RS. Reference ranges for testosterone in men generated using liquid chromatography-tandem mass spectrometry in a community-based sample of healthy nonobese young men in the Framingham Heart Study and applied to three geographically distinct cohorts. J Clin Endocrinol Metab. 2011;96(8):2430-2439.

49. Montanini V, Simoni M, Chiossi G, Baraghini GF, Velardo A, Baraldi E, Marrama P. Age-related changes in plasma dehydroepiandrosterone sulphate, cortisol, testosterone and free testosterone circadian rhythms in adult men. Horm Res. 1988;29(1):1-6.

50. Orwoll ES, Nielson CM, Labrie F, Barrett-Connor E, Cauley JA, Cummings SR, Ensrud K, Karlsson M, Lau E, Leung PC, Lunggren O, Mellstrom D, Patrick AL, Stefanick ML, Nakamura K, Yoshimura N, Zmuda J, Vandenput L, Ohlsson C, Osteoporotic Fractures in Men Research G. Evidence for geographical and racial variation in serum sex steroid levels in older men. J Clin Endocrinol Metab. 2010;95(10):E151-160.

51. Litman HJ, Bhasin S, Link CL, Araujo AB, McKinlay JB. Serum androgen levels in black, Hispanic, and white men. J Clin Endocrinol Metab. 2006;91(11):4326-4334.

52. Fiers T, Wu F, Moghetti P, Vanderschueren D, Lapauw B, Kaufman JM. Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis. J Clin Endocrinol Metab. 2018;103(6):2167- 2174.

53. Diagnostics Q. https://testdirectoryquestdiagnosti...osterone-free-dialysis-and-total-ms?cc=MASTER. 2022.

54. Laboratory MC. https://wwwmayocliniclabscom/testcatalog/Overview/83686#Clinical-and-Interpretive. 2022.

 

[madman]

If it went over anyone's head!

Hint (references)!


Jasuja 2022 (https://onlinelibrary.wiley.com/doi/abs/10.1111/andr.13310)

*Equilibrium dialysis was performed in 96-well plates (Harvard Apparatus, Holliston, MA) with semi-permeable membranes that allow species less than 10 kDa to pass through (41,43)


41. Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R. A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol. 2015;399:190-200.

43. Jasuja R, Spencer D, Jayaraj A, Peng L, Krishna M, Lawney B, Patel P, Jayaram B, Thayer KM, Beveridge DL, Bhasin S. Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states. iScience. 2021;24(6):102414



*Our normative ranges are similar to those reported by pioneers of this field using the legacy method (2) but the percent free testosterone in our reference sample differs from that reported by another laboratory (10,52); this difference in percent free testosterone from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods (10,52). The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time (53,54); because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.


10. Vermeulen A, Kaufman JM, Giagulli VA. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab. 1996;81(5):1821-1826.

52. Fiers T, Wu F, Moghetti P, Vanderschueren D, Lapauw B, Kaufman JM. Reassessing Free-Testosterone Calculation by Liquid Chromatography-Tandem Mass Spectrometry Direct Equilibrium Dialysis. J Clin Endocrinol Metab. 2018;103(6):2167- 2174

 

[tareload]

If it went over anyone's head!

Hint (references)!

I'd like to read the discussion and methods sections again with the ref list in hand.

Nah. Didn't go over my head and you have to put something out there to begin with in order for it to go over someone's head. Next time include the references with the paper text for the grown ups who may want to read the Discussion in context.

Thanks for the update now including the references so a complete review of the discussion and methods can be done with relevant context.

And by the way, cfTV still dominating from a practical model fitness perspective once you consider degrees of freedom vs Tru-T [see above in case you missed it or it went over your head ;-) ].

Jumping to conclusions? No. Trying to make sense of this and get to some conclusions. It is 2022 and I only have so much time on Earth.

Thanks again for sharing and fun to see cfTV/cfTV,Rev still kicking butt. For practical prediction of fT, go Team Vermeulen and its constitutive framework (easily shiftable with 1 DOF) and residuals plot far superior to cfTZ. Not even close.

 

[madman]

Nah. Didn't go over my head and you have to put something out there to begin with in order for it to go over someone's head. Next time include the references with the paper text for the grown ups who may want to read the Discussion in context.

Thanks for the update now including the references so a complete review of the discussion and methods can be done with relevant context.

And by the way, cfTV still dominating from a practical model fitness perspective once you consider degrees of freedom vs Tru-T [see above in case you missed it or it went over your head ;-) ].

Jumping to conclusions? No. Trying to make sense of this and get to some conclusions. It is 2022 and I only have so much time on Earth.

Thanks again for sharing and fun to see cfTV/cfTV,Rev still kicking butt. For practical prediction of fT, go Team Vermeulen and its constitutive framework (easily shiftable with 1 DOF) and residuals plot far superior to cfTZ. Not even close.

Better yet fetch them yourself next time before everyone starts running their mouths!

This is where it stands as of now!

How Accurate Is The Online Tru-T Free Test. Calculator?


Yet 4 years since the Fiers study let alone the most recent data which they have been sitting on and Bhasin continues to hammer this home!


Bhasin:

*the Vermeulen equation and others that assume a fixed Kd are just conceptually wrong and our data show it clearly that this model is completely wrong

*so the Vermeulen model or most of the linear equations underestimate free testosterone concentrations and the ensemble allosteric model (EAM) based upon our experimental data matches total testosterone within the range in which we have validated this

*Recent studies using modern biophysical techniques have shown that the binding of testosterone and estradiol to SHBG is a dynamic nonlinear process that involves an allosteric interaction between the 2 SHBG monomers, such that the Kd varies dynamically across the range of sex hormone and SHBG concentrations




Remember what I said about everyone and their brother waiting on the results/data from Phase II which still is ongoing?

Aim 3 who knew?


*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




He has not changed his stance and is still pushing the newer EAM (cFTZ/TruTTM)!

What's Coming? Accurate measurement of total and free testosterone levels for the diagnosis of androgen disorders

Accurate measurement of total and free testosterone levels for the diagnosis of androgen disorders (7/2022) Ezgi Caliskan Guzelce, MD, Clinical/ Research Fellow, Francesca Galbiati, MD, Fellow, Anna L. Goldman, MD, Assistant Professor of Medicine, Arijeet K. Gattu, MD, Fellow, Shehzad Basaria...

www.excelmale.com

*Testosterone levels should be measured preferably in a CDCcertified laboratory using validated assays; in cases of equivocal TT concentration and/or abnormal SHBG levels, free testosterone levels should be measured using equilibrium dialysis or calculated using an equation based on our current understanding of the dynamics of testosterone binding, such as the ensemble allostery model

Improving Diagnostic Accuracy and Treatment Decisions in Men with Hypogonadism

www.excelmale.com

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

*Free testosterone concentration is ideally measured using the equilibrium dialysis method, performed under standardized conditions.1,31 Direct tracer analog methods for measuring free testosterone concentrations are inaccurate, and therefore, their use is not recommended.35 Although several equations to estimate free testosterone concentration from total testosterone, SHBG, and albumin concentrations have been published,36-38 the estimation of free testosterone concentration performed using these equations are predicated upon accurate measurements of total testosterone, SHBG, and albumin concentrations.31,35 Furthermore, equations that are based on a linear model of testosterone’s binding to SHBG assume a fixed binding affinity (approximately 1 nM)31 and ignore the competing presence of other sex steroids, such as dihydrotestosterone and estradiol.

*Recent studies using modern biophysical techniques have suggested that the binding of testosterone and estradiol to an SHBG dimer is a dynamic process that involves allosteric interactions between binding sites on each of the 2 SHBG monomers such that the binding affinities of the 2 sites are not equivalent.36,39 The binding of a ligand to the first monomer influences the conformational and energetic states of both the monomers.39 The estimation of free testosterone concentration based on an ensemble allosteric model provides a close approximation of concentrations measured using equilibrium dialysis36

*the computations of free testosterone concentrations using the ensemble allostery model can be obtained at TruT Free Testosterone Calculator by FPT. Because of dynamic changes in the binding affinity of SHBG upon ligand binding, depending on the ligand and SHBG concentrations, no equation can accurately estimate free testosterone concentration under all conditions.39

 

[tareload]

Aim 3 who knew?


*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

You keep repeating the same stuff that is misleading as I have told you previously.

"if you repeat a lie often enough it becomes the truth"

Is this your goal?

How can phase 1 be complete until equilibrium dialysis method for fT has been standardized? Cherry pick some fT data anyone?

Great they can fit the binding parameters but Tru-T sucks at fitting the majority of clinical fT data available to date. Theory vs practice, etc.

Now if you don't want me commenting or analyzing data/papers in your threads I can start my own. I surely don't want to impede all the great material you share on here. As I have stated many times I am very appreciative of your efforts.

Who is everybody? Right now it is just me running my mouth. Don't you want interested educated people actively engaged in your threads discussing the science? Maybe you prefer being a big fish in a tiny pond? I'll defer to your preference. Just let me know.

Thought about lowering the dose a smidge there BRUH? May help with your bedside manner.

 

[tareload]

"Cargo Cult Science" - by Richard Feynman

...
That is the idea that we all hope you have learned in studying science in school--we never say explicitly what this is, but just hope that you catch on by all the examples of scientific investigation. It is interesting, therefore, to bring it out now and speak of it explicitly. It's a kind of scientific integrity, a principle of scientific thought that corresponds to a kind of utter honesty--a kind of leaning over backwards. For example, if you're doing an experiment, you should report everything that you think might make it invalid--not only what you think is right about it: other causes that could possibly explain your results; and things you thought of that you've eliminated by some other experiment, and how they worked--to make sure the other fellow can tell they have been eliminated.

Details that could throw doubt on your interpretation must be given, if you know them. You must do the best you can--if you know anything at all wrong, or possibly wrong--to explain it. If you make a theory, for example, and advertise it, or put it out, then you must also put down all the facts that disagree with it, as well as those that agree with it. There is also a more subtle problem. When you have put a lot of ideas together to make an elaborate theory, you want to make sure, when explaining what it fits, that those things it fits are not just the things that gave you the idea for the theory; but that the finished theory makes something else come out right, in addition.

In summary, the idea is to give all of the information to help others to judge the value of your contribution; not just the information that leads to judgement in one particular direction or another.

...
I would like to add something that's not essential to the science, but something I kind of believe, which is that you should not fool the layman when you're talking as a scientist. I am not trying to tell you what to do about cheating on your wife, or fooling your girlfriend, or something like that, when you're not trying to be a scientist, but just trying to be an ordinary human being. We'll leave those problems up to you and your rabbi. I'm talking about a specific, extra type of integrity that is not lying, but bending over backwards to show how you're maybe wrong, that you ought to have when acting as a scientist. And this is our responsibility as scientists, certainly to other scientists, and I think to laymen.

For example, I was a little surprised when I was talking to a friend who was going to go on the radio. He does work on cosmology and astronomy, and he wondered how he would explain what the applications of his work were. "Well," I said, "there aren't any." He said, "Yes, but then we won't get support for more research of this kind." I think that's kind of dishonest. If you're representing yourself as a scientist, then you should explain to the layman what you're doing-- and if they don't support you under those circumstances, then that's their decision.

One example of the principle is this: If you've made up your mind to test a theory, or you want to explain some idea, you should always decide to publish it whichever way it comes out. If we only publish results of a certain kind, we can make the argument look good. We must publish BOTH kinds of results.

...

 

[tareload]

"Cargo Cult Science" - by Richard Feynman

...
I would like to add something that's not essential to the science, but something I kind of believe, which is that you should not fool the layman when you're talking as a scientist. I am not trying to tell you what to do about cheating on your wife, or fooling your girlfriend, or something like that, when you're not trying to be a scientist, but just trying to be an ordinary human being. We'll leave those problems up to you and your rabbi. I'm talking about a specific, extra type of integrity that is not lying, but bending over backwards to show how you're maybe wrong, that you ought to have when acting as a scientist. And this is our responsibility as scientists, certainly to other scientists, and I think to laymen.

For example, I was a little surprised when I was talking to a friend who was going to go on the radio. He does work on cosmology and astronomy, and he wondered how he would explain what the applications of his work were. "Well," I said, "there aren't any." He said, "Yes, but then we won't get support for more research of this kind." I think that's kind of dishonest. If you're representing yourself as a scientist, then you should explain to the layman what you're doing-- and if they don't support you under those circumstances, then that's their decision.

One example of the principle is this: If you've made up your mind to test a theory, or you want to explain some idea, you should always decide to publish it whichever way it comes out. If we only publish results of a certain kind, we can make the argument look good. We must publish BOTH kinds of results.

...
Other kinds of errors are more characteristic of poor science. When I was at Cornell, I often talked to the people in the psychology department. One of the students told me she wanted to do an experiment that went something like this--it had been found by others that under certain circumstances, X, rats did something, A. She was curious as to whether, if she changed the circumstances to Y, they would still do A. So her proposal was to do the experiment under circumstances Y and see if they still did A.

I explained to her that it was necessary first to repeat in her laboratory the experiment of the other person--to do it under condition X to see if she could also get result A, and then change to Y and see if A changed. Then she would know the the real difference was the thing she thought she had under control.

She was very delighted with this new idea, and went to her professor. And his reply was, no, you cannot do that, because the experiment has already been done and you would be wasting time. This was in about 1947 or so, and it seems to have been the general policy then to not try to repeat psychological experiments, but only to change the conditions and see what happened.

Nowadays, there's a certain danger of the same thing happening, even in the famous field of physics. I was shocked to hear of an experiment being done at the big accelerator at the National Accelerator Laboratory, where a person used deuterium. In order to compare his heavy hydrogen results to what might happen with light hydrogen, he had to use data from someone else's experiment on light hydrogen, which was done on different apparatus. When asked why, he said it was because he couldn't get time on the program (because there's so little time and it's such expensive apparatus) to do the experiment with light hydrogen on this apparatus because there wouldn't be any new result. And so the men in charge of programs at NAL are so anxious for new results, in order to get more money to keep the thing going for public relations purposes, they are destroying--possibly--the value of the experiments themselves, which is the whole purpose of the thing. It is often hard for the experimenters there to complete their work as their scientific integrity demands.

...

 

[madman]

You keep repeating the same stuff that is misleading as I have told you previously.

"if you repeat a lie often enough it becomes the truth"

Is this your goal?

How can phase 1 be complete until equilibrium dialysis method for fT has been standardized? Cherry pick some fT data anyone?

Great they can fit the binding parameters but Tru-T sucks at fitting the majority of clinical fT data available to date. Theory vs practice, etc.

Now if you don't want me commenting or analyzing data/papers in your threads I can start my own. I surely don't want to impede all the great material you share on here. As I have stated many times I am very appreciative of your efforts.

Who is everybody? Right now it is just me running my mouth. Don't you want interested educated people actively engaged in your threads discussing the science? Maybe you prefer being a big fish in a tiny pond? I'll defer to your preference. Just let me know.

Thought about lowering the dose a smidge there BRUH? May help with your bedside manner.

You do realize that the ED device used in Phase 1 (SHBG:T binding) was the same ED device used in Ravi's current study?

Remember what I said about waiting on the completion of Phase II?


Aim 3 who knew?

*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




As I already stated in a previous thread.

No one should be using/relying upon the newer EAM (TruT™/cFTZ)
as Phase II is still ongoing and all of the results/data have not come out yet.

There has been an ongoing effort behind the scenes to provide further proof of the validity of the TruT™algorithm (cFTZ).

Everyone is still waiting!

This has to be done.

Having a hard time understanding why he is still pushing the newer EAM seeing as they have only completed Phase I and have not provided any further proof of the validity.

In one of his more recent lectures (3/2022) and papers (7/2022), he makes it clear where he stands and is still pushing the newer EAM.

Definitely has not changed his stance.

Seems odd as they would have been sitting on the data I posted here from their most recent paper.

Is there something they are sitting on?

Your guess is as good as mine!

Again we all very well know that when it comes to using any of the calculators for estimating free testosterone cFTV has been the go-to.

Already been validated (using assays for TT/SHBG no longer available)/re-validated in 2018 against current state-of-the-art methods.

Been in use for over 2 decades now.

Fiers camp let alone the CDC and many in the medical community is waiting to see how this unfolds.

The CDC is working hard behind the scenes on harmonized/standardization of free testosterone.

Still going to be a year or two before this s**t show comes to an end!




My reply from a previous thread:

This is coming from Fier's camp's most recent paper (posted in the link above).

They clearly state for the time being, as they are well aware there is still much going on behind the scenes.

Far from over!

*In contrast we concluded that, although overestimating free T by 20–30%, the formula according to Vermeulen was independent of SHBG, total T, and albumin [221]. Taken into consideration the moderate bias level, but most importantly less influenced by the latter three variables, the Vermeulen formula, for the time being, still appears to be the most robust approximation and deserves our recommendation as a free T calculator for clinical use








Even then hate to burst your bubble Lil man but highly doubt cFTV let alone cFTZ will come out on top.

Remember what I said before or did everyone forget?


*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


Wonder why the f**k they would go and do that!






Ravi 10/2022

This study also has some limitations. These reference ranges were derived from single morning samples, which discount the pulsatile and diurnal secretory rhythms. Previous analyses show that early morning testosterone levels, obtained in a manner similar to that used by physicians in practice, are associated cross-sectionally with symptoms and clinical outcomes (38-40,48). We report reference ranges in men <40 years of age but also provide the distribution of free testosterone levels by age groups. This approach of generating the reference range in healthy young men is analogous to the use of T-scores for bone mineral density. Although the sample included men of various races and ethnicities, the number of nonwhites was not large enough to offer sufficient power to detect meaningful differences in free testosterone levels among racial or ethnic subgroups. The data on geographic and racial differences in total testosterone levels are inconsistent (50,51), and no study has examined racial and ethnic differences in free testosterone levels using equilibrium dialysis in different geographic regions of the world. Additional investigations of multi-ethnic cohorts to evaluate the generalizability of the proposed reference limits to men of other races and ethnicities in different regions of the world are important. Although the sample size was within the IFCC guidelines for analytes with normal distribution, there were relatively small numbers of men within each decade of age and a larger sample size would provide more robust estimates of the reference ranges by age decades.

Our normative ranges are similar to those reported by pioneers of this field using the legacy method (2) but the percent free testosterone in our reference sample differs from that reported by another laboratory (10,52); this difference in percent free testosterone from that reported by another research laboratory (<2.8%) could be due to differences in buffer composition or in other assay conditions that are not apparent in the published methods (10,52). The normative ranges of most commercial laboratories have changed substantially in recent years suggesting changes in their procedures over time (53,54); because procedures used by the commercial laboratories and the details of how reference ranges were derived are not published, an evaluation of these procedures was not feasible.

These reference ranges, generated in a reference sample of healthy men, should not be applied to other assays in other laboratories without appropriate cross-calibration of assays. Differences in study populations, time of sample collection, and testosterone assays can contribute to the differences in reference ranges. The adoption of a standardized procedure for measuring free testosterone and cross-calibration of the testosterone assays against an accuracy-based benchmark such as the CDC's HoST program will facilitate the application of these reference ranges across laboratories

The data here defines reference intervals from a population of healthy nonobese men using a standardized equilibrium dialysis procedure coupled to a HoST-certified LC-MS/MS assay and represent an important first step. Further studies are needed to determine how well these reference limits can be applied to the diagnosis of androgen deficiency in clinical populations and in people of different races and ethnicities in different geographic regions. The association of low free testosterone defined using these criteria with incident outcomes in epidemiologic studies should be studied. Importantly, randomized trials are needed to determine whether testosterone therapy improves outcomes in men, who have free testosterone below these reference limits.