Quest Equilibrium dialysis test - free T seems low?

[Andy2019]

Hello,

First post, but long time follower. Really appreciate the knowledge I've obtained from this forum. I know there are many posts on this but none seem to exactly characterize my direct quadrium. Hope for some assistance and good conversation. Apologies about any retreading on topic..

140mg test enth week (EOD dosing)
Last labs Quest
Total T, MS: 1261 ng/dl
Testosterone, Free (EqDial): 196 pg/ml
SHBG: 37
Albumin: 4.0


So my question is regarding the free t results from quest using their equilibrium dialysis test. It appears low given the SHBG level and normal Albumin. When using Free Testosterone Calculator — with Bioavailable Levels online calculator (Vermeulen) and using same inputs, my free test comes back at 32ng/dl, which is more than 50% higher than the quest results. I take meticulous notes on how I am feeling on different protocols and want to pair with labs for analytical comparison. Happy to comment on how I feel (which is pretty good) but more was hoping to get a read on where some experienced members feel my free t is truly sitting.

Thanks!

 

[Andy2019]

Also apologies for not clarifying - I know there are numerous posts on the difference in methods. My main confusion is in the free t number given my higher total t and pretty normal shbg of 37. From scanning dozens of other threads with labs I would expect with my shbg to achieve these free t levels with much lower total total t. For comparison, using the same online calculator, a total t of 900 achieves the same levels that quest e.d. shows at my levels of almost 1300 tt.. Thanks

 

[Systemlord]

From scanning dozens of other threads with labs I would expect with my shbg to achieve these free t levels with much lower total total t.

You make the assumption that the binding affinity for SHBG is the same person to person. If you look at individual T receptors, you'll see differences in receptor density and sensitivity.

 

[Andy2019]

Thanks for responding. Guessing by this response you would favor the equilibrium dialysis method, which presumably accounts for this.. Does this make the calculated levels the less accurate of the two, and more of an approximation than actual measurement? Why so much "Vermeulen" love on here?

 

[madman]

Also apologies for not clarifying - I know there are numerous posts on the difference in methods. My main confusion is in the free t number given my higher total t and pretty normal shbg of 37. From scanning dozens of other threads with labs I would expect with my shbg to achieve these free t levels with much lower total total t. For comparison, using the same online calculator, a total t of 900 achieves the same levels that quest e.d. shows at my levels of almost 1300 tt.. Thanks

No way in hell your FT is lowish let alone subpar with a whopping TT 1261 ng/dL and normal SHBG.

Your FT 19.6 ng/dL based off that specific assay/reference range is high!

Even when using the cFTV your FT is well over the top-end.

Also keep in mind as of now cFTV tends to overestimate slightly when compared against a standardized Equilibrium Dialysis assay.

As I have stated in previous threads Quest/Labcorp let alone any of the other labs Equilibrium Dialysis or Equilibrium Ultrafiltration assays in the US or any other country are not standardized!

Quests ED assay reference range is 35-155 pg/mL and your results are over the top-end of the reference range and well over where a healthy young male would be using the same assay.

Your FT 19.6 ng/dL is high!

Forget getting caught up in the different reference ranges for the same assays whether (ED or UF) used by different labs let alone trying to compare the results of ED vs UF, or ED/UF vs the cFT methods.

Test using the same lab/same assay (most accurate).

Compare your blood work using the same lab/same assay (most accurate).

If you choose to use/rely upon the cFT methods than stick with it.

Keep in in mind the calculated methods even have flaws.

We need accurate and standardized free testosterone assays with harmonized reference ranges!

This is key!

* 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

*The lack of standardization of the equilibrium dialysis method among laboratories has been a barrier to the generation of a harmonized reference range for free testosterone levels; until such rigorously-derived harmonized reference ranges become available, the clinicians currently must rely on reference ranges provided by a laboratory or those published from the analyses of large epidemiologic studies

AGE SPECIFIC CALCULATED FREE TESTOSTERONE RANGES IN ADULT MEN

INTRODUCTION AND OBJECTIVE Measurement of free testosterone (FT) levels can be an important step in the evaluation of men with suspected testosterone deficiency. While it is well known that FT levels decline with age, its clinical utility is limited due to the lack of data on age specific...

www.excelmale.com

Take home points:

*Assays that are standardized are designed to provide accurate results, traceable to “true” value-assigned certified reference materials and gold-standard reference methods. Results obtained using standardized methods can be compared across assays, institutions, populations, and past and future test results, thereby improving diagnosis, treatment, and outcomes of patients

* 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

 

[madman]

Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods

Only at Excel, are we gonna take you deep! Clinical Utility and Analytical Aspects of Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods (2022) Mark M. Kushnir, Heather A. Nelson, and Kelly Doyle Background The free hormone (FH) hypothesis states that hormone...

www.excelmale.com

 

[Andy2019]

Thanks for the follow up and agree with using same method. Not trying to call you out but saw this on another thread from you -
"Most men do well having a FT level in the 30 ng/dL range.....some run higher levels and others lower"
I'm interested in trying to "experiment" with getting my free t in the 25/30ng/dl as there seems to be a lot of anecdotes of feeling optimized there. I see a lot of people there on this forum with <1000TT which concerned me that I would likely need ~1500-1800 to get there using same testing method.

 

[Andy2019]

Thanks for responding. Guessing by this response you would favor the equilibrium dialysis method, which presumably accounts for this.. Does this make the calculated levels the less accurate of the two, and more of an approximation than actual measurement? Why so much "Vermeulen" love on here?

would total t levels like mine scare you away from trying to creep dose up?

 

[madman]

Thanks for the follow up and agree with using same method. Not trying to call you out but saw this on another thread from you -
"Most men do well having a FT level in the 30 ng/dL range.....some run higher levels and others lower"
I'm interested in trying to "experiment" with getting my free t in the 25/30ng/dl as there seems to be a lot of anecdotes of feeling optimized there. I see a lot of people there on this forum with <1000TT which concerned me that I would likely need ~1500-1800 to get there using same testing method.

This would be based on the EAM (ensemble allosteric model) cFTZ which is still in development!

As I stated previously cFTV tends to overestimate slightly when compared to a standardized Equilibrium Dialysis assay.

Again with a whopping TT 1261 ng/dL and normal SHBG your FT is high (Quest ED assay/cFTV).

Your FT level is well over where a healthy young male in his prime would be!

Keep in mind your peak TT, FT and estradiol will be higher if your blood work was done at the true trough (48 hrs post-injection) seeing as you are injecting EOD!

 

[madman]

You make the assumption that the binding affinity for SHBG is the same person to person. If you look at individual T receptors, you'll see differences in receptor density and sensitivity.

Some key takeaways here!


*wild type SHBG is present in nearly 98% of Caucasians

*Genetic polymorphisms suggested to affect SHBG concentration or steroid-hormone binding affinity are relatively rare in this population-based cohort of healthy men

*In these men, analyzed SNPs were relatively prevalent and affected serum concentrations of total T and SHBG but not calculated or measured free T except for a higher trend in rs6259 homozygotes.

US Patent Application for METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF SEX HORMONE DISORDERS Patent Application (Application #20220206018 issued June 30, 2022) - Justia Patents Search

The technology described herein is directed to the diagnosis and treatment of sex hormone disorders and/or deficiencies, such as estrogen and/or testosterone disorders and/or deficiencies.

patents.justia.com

EAM (ensemble allosteric model) cFTZ

[0325] The current algorithm and the experimental data reported here were generated using wild type SHBG which is present in nearly 98% of Caucasians. Genome wide association studies have revealed several SHBG polymorphisms, two of which have been reported to affect testosterone binding to SHBG (28). Therefore, in future, the algorithms may include a term for the SHBG genotype.

SHBG Gene Polymorphisms and Their Influence on Serum SHBG, Total and Free Testosterone Concentrations in Men

AbstractContext. Genetic variation in SHBG structure may affect estimates of sex steroid exposure by altering the affinity of the protein for its ligand. C

academic.oup.com

Abstract

Context

Genetic variation in sex hormone-binding globulin (SHBG) structure may affect estimates of sex steroid exposure by altering the affinity of the protein for its ligand. Consequently, free hormone calculations assuming constant binding affinity may, for certain genetic variations, lead to incorrect diagnoses if genetic variation is not taken into consideration.


Objective

To investigate the effects of genetic variation in SHBG on calculated and measured serum free testosterone (T) in men.


Design, setting and participants

Population-based sibling-pair study in 999 healthy men aged 25 to 45 (mean: 34.5) years.


Main outcome measures

Genotyping using microarray (Illumina®) for SNPs suggested to affect binding affinity and/or concentration of SHBG or T. SHBG concentrations were measured using immunoassay and in a subset (n = 32) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total T was measured using LC-MS/MS. Free T was calculated and in a subset (n = 314) measured directly using LC-MS/MS after equilibrium dialysis.


Results

Allelic frequencies of analyzed SNPs ranged from 0.5% to 58.2%. Compared to wild-type, SHBG concentrations were lower in rs6258 heterozygotes (-24.7%; p < 0.05) and higher in rs6259 heterozygotes, rs727428 homozygotes, and carriers of rs1799941 (+10.8 to 23.1%; all p < 0.05). Total T was higher in rs727428 homozygotes and carriers of rs5934505, rs1799941 and rs6259 (+3.9 to 21.4%; all p < 0.05). No clear effects on measured free T were found, except for a trend towards higher values in rs6259 homozygotes, significant for calculated free T (+18.7%; p < 0.05) in the larger global study population.


Conclusion

In these men, analyzed SNPs were relatively prevalent and affected serum concentrations of total T and SHBG but not calculated or measured free T except for a higher trend in rs6259 homozygotes.

Binding affinity affecting SHBG SNPs do not majorly affect calculated estimates of free testosterone | ECE2022 | European Congress of Endocrinology 2022 | Endocrine Abstracts

www.endocrine-abstracts.org

Conclusion

Genetic polymorphisms suggested to affect SHBG concentration or steroid-hormone binding affinity are relatively rare in this population-based cohort of healthy men. Although carriers did present with different SHBG levels and free T-ratios compared to non-carriers, our results do not disprove the calculators’ accuracy in men heterozygous for rs6258 or rs6259, despite in vitro experiments suggesting a 1.8 reduced binding affinity and reduced clearance rate respectively compared to wild-type SHBG.