Chemistry, toxicology & urinalysis

General

• The 2 principal thyroid hormones are thyroxine (also known as T4 or L-3,5,3',5'-tetraiodothyronine) and triiodothyronine (T3 or L-3,5,3'-triiodothyronine) (Ento Key: Thyroid Physiology and Thyroid Function Testing [Accessed 25 June 2020])
  
  

• Thyroxine (T4)
  • Usual daily production is 110 nmol/day; half life is 5 - 7 days
  • 100% produced in thyroid
  • 50% of T4 is 5' deiodinated to form T3, 40% is 5 deiodinated to form reverse T3 via 5-deiodinase (Ento Key: Thyroid Physiology and Thyroid Function Testing [Accessed 25 June 2020])
    
    

• Triiodothyronine (T3)
  • Usual daily production is 50 nmol/day; half life is 1 day
  • 80% from deiodination of T4 in nonthyroid tissue, 20% arises in thyroid gland
• Most of the TH circulating in the blood is bound to TH binding proteins: thyroxine binding globulin (TBG), transthyretin (TTR) and albumin
• Only a very small fraction of unbound TH (0.03% of T4 and 0.3% of T3) is biologically active and signal all tissues by binding TH nuclear receptor (THR)
• TH is metabolized by deiodination, conjugation with glucuronide and sulfate, ether link cleavage and side chain modification (oxidative deamination and decarboxylation) (Endotext: Metabolism of Thyroid Hormone [Accessed 25 June 2020])
  
  

• Reverse T3 (rT3)
  • Also known as 3,3',5'-triiodothyronine
  • Biologically inactive metabolite of T4 formed by selective deiodination
  • Not routinely measured
  • Serum rT3 levels are increased in systemic nonthyroidal illnesses except chronic renal failure and in patients taking drugs such as amiodarone, propylthiouracil, dexamethasone, propranolol and ipodate

• Free T4 measurement is preferred to total T4 whereas total T3 is preferred to free T3 (Best Pract Res Clin Endocrinol Metab 2013;27:689)
• Presence of factors in serum that affect free TH measurement include:
  • Interferences (T4/T3 antibodies, human anti-animal antibodies) (Endocr Rev 2018;39:830)
  • Variant thyroid hormone binding proteins: dominantly inherited genetic variants of albumin (familial dysalbuminemic hyperthyroxinemia [FDH] or transthyretin [transthyretin associated hyperthyroxinemia (TTR-AH)])
  • Drugs (e.g. heparin and aspirin) (N Engl J Med 2019;381:749)

Methods of free TH measurement

• Free T4 index
  • Free T4 index is calculated by dividing the total T4 by the T3 uptake ratio (percentage)
  • T3 uptake test is performed by incubating the patient's serum with radiolabeled T3 tracer and subsequently adding resin that traps the remaining unbound radiolabeled T3; the value reported is the percent of tracer bound to the resin, which varies inversely with the number of available free binding sites for T3
  • T3 uptake was designed to distinguish TBG excess and deficiency from hyperthyroidism and hypothyroidism:
    • Hyperthyroidism: high serum total T4, high T3 uptake, high free T4 index
    • TBG excess: high serum total T4, low T3 uptake, normal free T4 index
    • Hypothyroidism: low serum total T4, low T3 uptake, low free T4 index
    • TBG deficiency: low serum total T4, high T3 uptake, normal free T4 index
• Free TH ligand assays (indirect method) (Biochemia Medica 2016;26:436)
  • Competition immunoassay is used
    • Serum TH compete with labeled TH analog for a fixed number of TH antibody binding sites
    • Serum TH compete with an immobilized TH analog for a labeled TH antibody
  • Free hormone concentration is inversely proportional to the signal generated with fluorescence or chemiluminescence
  • Different immunoassay platforms are available
    
    

    • 1 step labeled T4 analog (Immulite, Siemens)
    • 1 step analog 2 step incubation (Cobas, Roche Diagnostics)
    • 2 step labeled T4 analog method (DXi, Beckman Coulter, Architect, Abbott)
    • 1 step labeled antibody method (Vitros, Ortho Clinical Diagnostics)
  • 2 step method was less susceptible to T4/3 autoantibodies than the 1 step method
• Ultrafiltration (UF) and equilibrium dialysis (ED) (direct method) (Best Pract Res Clin Endocrinol Metab 2013;27:689)
  • Methods with physical separation against ultrafiltrate / dialysate membrane and direct measurement of free T4 in protein depleted serum by immunoassay or LC-MS/MS
    
    

  • Not affected by changes in binding protein concentration, T4/T3 autoantibodies or nonthyroidal illness
  • Free thyroid hormone concentrations measured by LC-MS/MS correlate to a greater degree with log TSH values compared to concentrations measured by immunoassay (Clin Chem 2009;55:1380)
  • Time consuming, expensive and requires rigorous control of the pH, temperature, membrane type / cutoff, minimum dilution and dialysis buffer

Reference value

• Depends on different platforms and methods
• Cobas, Roche Diagnostics
  • FT4 12 - 22 pmol/L (0.93 - 1.7 ng/dL)
  • FT3 3.1 - 6.8 pmol/L (2 - 4.4 pg/mL)

Conversion factor

• FT4: multiply by 12.87 to convert from ng/dL to pmol/L
• FT3: multiply by 1.54 to convert from pg/dL to pmol/L

A 65 year old man with no history of thyroid disease is admitted to the medical intensive care unit for septic shock. Thyroid function test is performed due to hyponatremia: TSH 5 μU/mL (0.3 - 4.1), free T4 0.9 ng/dL (0.8 - 1.8), free T3 1.2 pg/mL (1.6 - 4).

What is the most likely cause of abnormal thyroid function test in this patient?

1. Heterophile antibodies
2. Nonthyroidal illness
3. Primary hypothyroidism
4. Subclinical hypothyroidism
5. TSH producing pituitary adenoma

B. Nonthyroidal illness. TFT showed normal free T4, low T3 and slightly elevated TSH. Nonthyroidal illness is the likely background condition. In the early course of illness, free T3 will firstly decrease due to decrease of peripheral conversion while free T4 is still maintained; TSH level is variable (0.1 - 20 μU/mL). Heterophile antibodies is incorrect because normally heterophile antibodies will interfere with TSH measurement, causing falsely high TSH but normal free T4 and T3 level. Primary hypothyroidism is incorrect because free T4 level is normal and TSH level is too low for primary hypothyroidism. Subclinical hypothyroidism is incorrect because free T3 level is low. TSH producing pituitary adenoma is incorrect because free T3 and T4 level is not high. TFT pattern in TSH producing pituitary adenoma is high free T3/T4 and normal or high TSH.

Comment Here

Reference: Thyroid function panel

A 55 year old woman with weight gain and fatigue was found to have an elevated serum TSH and was started on levothyroxine (LT4). She was referred for assistance in managing thyroid replacement therapy. Physical examination showed a pulse rate of 80 beats per minute, no goiter or thyroidectomy scar and normal deep tendon reflexes.

Date	TSH (0.3 - 4.1 μU/mL)	FT4 (0.8 - 1.8 ng/dL)	Levothyroxine dose (mg/day)
June 2019	12	1.3	None
September 2019	11	1.7	0.075
December 2019	10.5	2.1	0.100

Which of the following is the most likely explanation for these findings?

1. Free T4 autoantibodies
2. Heterophile antibodies
3. Poor absorption of levothyroxine
4. Resistance to thyroid hormone
5. TSH producing pituitary adenoma

B. Heterophile antibodies. Normal free T4 and high TSH on 06/2019 were compatible with subclinical hypothyroidism. Differential diagnosis is NTI recovery phase, TSH resistance, assay interference (heterophile antibodies, macroTSH), poor absorption of levothyroxine. After trial of LT4 replacement TFT, there were high free T4 and high TSH (similar level as the initial TSH) levels. The most likely explanation is that the patient had heterophile antibodies that caused falsely elevated TSH. When the patients takes LT4, free T4 level is elevated but TSH still high at the same level because of heterophile antibodies. Free T4 autoantibodies is incorrect since free T4 level was normal at initial evaluation. Poor absorption of levothyroxine is incorrect since free T4 level increased after LT4 treatment. Resistance to thyroid hormone is incorrect since free T4 level was normal at presentation. TSH producing pituitary adenoma is incorrect since the patient did not have clinical thyrotoxicosis and free T4 level was normal at initial evaluation.

Comment Here

Reference: Thyroid function panel