Thyroid hormones in sport: use or abuse?

Code: 18D06XD 

Among the substances declared by the athletes during the sample collection sessions and reported in the doping control forms (DCF), it is quite frequent to observe supplements or medication based on thyroid hormones. This supplements or medications contain not only levothyroxine (T4) or triiodotironine (T3) but some of their derivatives as Triacana (3,5,3’-triiodothyroacetic acid) or Tetrac (3,5,3’,5’-tetraidothyroacetic acid). In 2017, at the WADA accredited antidoping laboratory of Rome, the athletes declaring to consume this kind of substances, was ten times higher compared to the prevalence of hypothyroidism in Italy. This high incidence of their consumption among the athletes in conjunction with their metabolic actions and the consequences of their intake over the health, impose to investigate which is the real use of these compounds and to start to investigate a new potential doping practice.

We plan to investigate the real analytical possibilities for determining the prevalence of use in sports using the available capabilities in the antidoping laboratories, ideally in already existing methods starting from urine or serum samples collected for other antidoping analyses.  This would allow, if considered convenient, to include an additional section in the athletes biological passport (ABP) endocrinological module for these hormones.

The main goal is to monitor thyroxine hormones (TSH, freeT3, freeT4 and freeT4/freeT3 ratio) in athlete’s serum and investigate the best biomarkers in urine focusing the attention in their potential inclusion in the ABP endocrinological module. Once the method developed and biomarkers chosen, the proposed approach will be applied to different thyroid conditions and the profiles under some administrations evaluated. 

Main findings

A method to detect thyroid hormones (TH) and some related metabolites in serum was validated using the LCMS/MS technique. The correlation of the validated method with RIA showed adequate results and one of the advantages of the LCMS technique with respect to RIA is that it does not show cross-interferences as is the case of T3 with triiodo-thyronacetic acid, which could happen with other metabolites. One of the disadvantages is the matrix effect, so it must be treated with particular care in the implementation process. Analysis of serum samples from athletes who reported levothyroxine consumption showed elevated levels of thyronine and tetraiodo-thyronacetic acid. At least preliminarily, these two compounds and the relationships that involve them (with T3 and T4) could be used as markers for the detection of levothyroxine consumption in serum.

Also, a method was validated for urine that allows the detection of THs and their metabolites. The method was applied to the total urine fraction (free + glucuronide conjugates + sulfate conjugates) but can be applied to the fractions separately. It was based on extraction at neutral pH to have a global idea of the excretion in urine of acidic (thyronacetic acids) and basic (thyronamines) metabolites.

Applying the validated method in LCMS to the total fraction of serum and urine, it was possible to verify that the levels of T3 and T4 of athletes are lower than those of a euthyroid population even when they are under the administration of TH supplements, coinciding with episodes of hypothyroidism described in reports focused

on high-performance sports. However, the high efficiency of the HTP axis (which proved to be, throughout the study, the most strict, refined, and exquisite) does not allow us to observe differences between athletes who do not declare and those who declare having used TH supplementation.

Direct measurements of T3 and T4 do not seem to be useful in these cases since everything seems to indicate that external supplementation aims to cover the needs of thyroid hormones to maintain homeostasis. Therefore, it could be assumed that the metabolic pathways observed in a euthyroid individual after the administration of TH are not the same as those observed in athletes. The detection of the administration of TH, at least triiodothyronine (T3) and levothyroxine (T4) in urine could work when dealing with euthyroid individuals, either by applying cut-off values of ratios (for T3) or the presence of T1 (for T4). In individuals with hypothyroidism where the tendency is towards the maintenance of homeostasis (as in the case of athletes), it was not possible to clearly detect the consumption.

Regarding to Endogenous Steroid Profile in urine, differences in the markers of the ABP steroidal module were found considering samples of athletes declaring and not declaring the supplementation with TH in the Doping Control Form. Although not all assessed parameters showed significant differences, there was observed a tendency to decrease steroid concentrations in the group of athletes who declared the consumption of levothyroxine. Female groups showed higher differences between declaring and not-declaring athletes' respect to males. The results for pregnanediol were a remarkable point, especially in the case of males. The information on TH therapy by athletes may be helpful for the correct interpretation of the ABP as happens for other markers considered confounding factors of the urinary steroid profile in the current WADA documents.