The sample consisted of 20 persons of both sexes with a diagnostic impression of possible hyperthyroidism and 20 persons with no underlying pathology (control group). The following criteria were taken into consideration for the selection of the sample:

a.    Inclusion criteria

1.      Persons of both sexes willing to participate in the study voluntarily.

2.      Over 18 years of age.

3.      Non-smokers and low or sporadic alcohol consumption.

b.   Exclusion criteria

4.      Individuals suffering from chronic or hematological diseases.

5.      Persons occupationally exposed to heavy metals.

Biological sample

Each individual participating in the study was took a specific urine sample (the first morning urine, before starting activities), in clean plastic containers, after indication for correct collection. Samples were refrigerated between 2 and 8°C and transported to the FITOQUIMICA20 C.A Laboratory.

For the extraction of blood samples, the rules of asepsis and antisepsis were followed. 10 mL of blood was extracted from the antecubital vein with a 12 mL disposable injector and a 21G x 1'' needle, then the contents of the injector were slowly deposited into two tubes previously identified with the patient's data. 5 mL of blood was placed in a tube with two drops of ethylenediaminetetraacetic acid (EDTA) for Pb analysis. The other 5 ml were intended for the analysis of hormones.

Determination of mercury

It was performed by method cold vapor atomic absorption spectrophotometry, using the method recommended by the National Institute for Occupational Safety and Health (NIOSH, 1994). To 4 mL of uncentrifuged urine, 7 mL of 65%  nitric acid (HNO₃) (Merck KGaA, Germany) was added. After 5 minutes, 60 mL of deionized water was added and, to reduce the mercury ion Hg²⁺ to its elemental form and initiate the emission of cold vapors, 1 mL of 20% SnCl₂ solution prepared from of SnCl₂•2H₂O ACS 98% (Sigma-Aldrich Co., USA). The absorbance measurement of the samples at 253.7 nm (maximum absorption at the mercury resonance line) was performed with a Bacharach® MAS-50B cold vapor spectrophotometer. A calibration line was also elaborated using a mercury chloride standard in a range of concentrations from 0.2 to 3 µg/dL.

 Creatinine determination

Creatinine analysis by the modified Jaffe method is based on reacting the sample with sodium picrate, in an alkaline medium, to form a red chromogen with an absorption maximum at 510 nm (Delanghe & Speeckaert, 2011). Analytical results are frequently expressed in micrograms of mercury per gram of creatinine. The method consists of diluting the urine sample with distilled water (1/100) to a final volume of 5 mL. An aliquot of 0.5 mL of sample was taken, 0.5 mL of distilled water and 2 mL of alkaline picrate were added. The latter reagent was prepared by mixing 20 mL of a saturated aqueous solution of ACS 99% picric acid (Merck KGaA , Germany) and 4 mL of 10% NaOH ACS 97% (Sigma-Aldrich Co., USA).

Determination of lead by atomic absorption

Blood collected in polyethylene tubes with heparin as an anticoagulant is hemolyzed. Lead is complexed with ammonium pyrrolidinedithiocarbamate (APDC) and the complex formed is extracted with methyl isobutyl ketone (MIBK). The lead contained in the organic phase is determined by flame Atomic Absorption Spectrophotometry, at a wavelength of 283.3 nm, using a direct quantification method. To determine analyte concentrations in a sample, the absorbances of standard solutions or standards of known analyte concentrations were first determined (Frank et al., 2029).

 The value of these absorbances was then plotted against the concentrations, thus obtaining the calibration curve(concentration range from 0.1 to 10 µg/dL with an R² of 0.99 to validate the straight line). Generally, analyte concentrations that have a linear relationship with absorbance are used, becoming known as the absorbance/concentration relationship “Calibration line”. Once the calibration line was established, the readings were taken and the concentration of the analyte was obtained (Martínez, 2020).

Determination of TSH, T3 and free T4 in serum

The commercial brand chroma™ was used, which is a lateral flow chromatography fluorescence immunoassay (FIA) for the quantitative determination of the level of Thyroid Stimulating Hormone (TSH), Free T3 and Free T4 in serum or plasma.

Bioethical considerations

In order to adequately select participants, informed consent was obtained, where the objectives of the study were explained to the patients and volunteers. The research adhered to the criteria established in the fifth revision of the Declaration of Helsinki.

Statistical Analysis

Metals, creatinine and thyroid profile were analyzed in triplicate. A descriptive statistical analysis was performed using measures of dispersion and central tendency as mean and standard deviation. Likewise, association and comparison tests(t-Student and Pearson correlation) were applied using the statistical program, Statistix10.0 for Windows.