Lina Luo (Presenter)
Pfizer Inc
Bio: Lina Luo is working in the Bioanalytical Toxicology Group of Biomarkers with Pfizer Drug Safety R&D in Groton, CT. She joined Pfizer in 2004, since then she has been working in DSRD for 12 years supporting from early stage to post marketing toxicology studies. Lina possesses an extensive expertise in analytical methods such as HPLC and LC-MS/MS technologies, and has applied her analytical skills for the development of numerous safety biomarker assays in preclinical studies as well as in clinical trials.
Authorship: Lina Luo(1); Kate Rowatt (2); Jennifer Colangelo (1)
(1) Pfizer Global Research & Development, Drug Safety Research and Development, Groton, CT; (2) University of Connecticut, Storrs, CT2
| Short Abstract Compounds in drug development may enhance the metabolism or clearance of thyroid hormones in animal models, triggering a sequence of toxicity events, and accurate measurements of thyroid hormones are important to aid in understanding the response. In this project, we utilized a validated LC/MS/MS method to profile the five thyroid hormones (T4, T3, rT3, 3,3’-T2, and 3,5-T2) in serum samples from various animal species and humans. These results enabled us to establish the distribution in normal animals and evaluate the influence of different factors, such as gender, on the profile. |
Long Abstract
Introduction:
Regulating the levels of thyroxine (T4), triiodothyronine (T3), 3,5’,3’-triidothyronine (rT3), and diiodothyronine isomers is an integral component of thyroid hormone homeostasis, which is important in understanding metabolism and function of thyroid hormones at the cellular level. The thyroid hormones, T3 and T4, are tyrosine-based hormones produced by the thyroid gland. They are converted to their derivatives, 3,5’,3’-triidothyronine (rT3), 3,3’-diiodothyronine (3,3’-T2), and 3,5-diiodothyronine (3,5-T2), by selenodeiodinase enzymes. These hormones have important functions including the maintenance of metabolism, regulating the growth and development of the body and brain, as well as regulation of digestion, heart rate, and muscle function in humans and preclinical species.
The thyroid is also a major target organ of toxicity during drug development. Potential new drugs that enhance metabolism or clearance of thyroid hormones in animals often trigger a sequence of toxicity events, so the accurate measurement of thyroid hormones in preclinical toxicology studies and clinical trials is important. Liquid chromatography– tandem mass spectrometry (LC/MS/MS) is an emerging technique for the measurement of thyroid hormones in the clinical setting and is preferred over immunoassay methods. The ability to profile isomeric thyroid hormones is another advantage of the mass spectrometry method over immunoassay methods. Preclinically thyroid hormones are typically measured by immunoassays on multiple platforms based on species. We had successfully consolidated 10 different immunoassays to a single MS method by measuring serum T3 and T4 simultaneously for multiple preclinical species. In this project, we utilized a validated LC/MS/MS method to profile the five thyroid hormones, namely, T4, T3, rT3, 3,3’-T2, and 3,5-T2, to investigate the concentration ranges across different animal species and genders, and to assess thyroid hormone profiles in preclinical species and humans.
Methods:
Thyroid hormones were quantified by LC/MS/MS on a Waters Acquity UPLC system interfaced to an AB Sciex 5500 QTrap in positive ion mode using stable labeled internal standards (T3-13C6 and T4-d5). A 100uL aliquot of serum/plasma was deproteinized, and chromatographic separations were performed by gradient elution on a Restek Biphenyl column, 2.1 x 50 mm, 5 µm. Calibration standards ranging from 0.01 to 100 ng/mL and quality controls at 2, 10, and 50 ng/mL were prepared in charcoal stripped serum. Accuracy and precision were evaluated by analyzing three concentration levels of quality controls on three different days. A run was deemed acceptable if the QC samples were ±20% of the nominal concentrations and the coefficient of variance (CV) did not exceed 15%.
Application of the assay to various preclinical species and humans was conducted by measuring thyroid hormones in serum/plasma from normal mice, rats, dogs, monkeys, and humans. Both male and female samples from each preclinical species and humans were collected for gender comparison. To evaluate the effect of food and collection time on hormone levels in the dog, time course samples were collected on two different days (Days 7 and 14) immediately before food presentation and at 0.5, 1, 1, 2, 4, 6, and 24 hours after food presentation. Serum and plasma samples were also compared to evaluate matrix impact in dogs.
Results:
• Serum concentrations of five thyroid hormones were measured in normal mice, rats, dogs, monkeys and human samples; 3,3’-T2 and 3,5’-T2 levels were below the quantitation limit (LOQ) of 0.01 ng/mL in all preclinical species; 3,5-T2 levels in human was also below the LOQ of this assay.
• In all the serum samples tested, dogs had much lower T4 concentrations than any other species and or humans; however, the T3 and rT3 concentrations in dogs were equivalent to other species.
• No significant gender difference was observed on rT3 concentrations for all samples; however, T4 showed significant gender difference in all preclinical species and humans, and T3 showed significant difference between male and female Sprague-Dawley rats.
• In the dog samples, all thyroid hormones exhibited a similar trend over a 24 hr time course, also similar changes on different sample collecting times and dates.
• No significant difference was observed between serum and plasma in samples collected from dogs.
• The ratio of T3 to rT3 was above 2 in all samples; the monkeys had higher T3/rT3 ratio compared with other preclinical species and humans.
Conclusions:
• The LC/MS method provides the specificity, precision, and sensitivity necessary for the reliable measurement of T4, T3, rT3, 3,3’-T2, and 3,5-T2 simultaneously.
• Serum thyroid hormones were profiled, and preliminary reference ranges were generated in mice, rats, dogs, monkeys, and humans.
• Statistically significant differences between male and female groups were observed for T4 levels in all preclinical species and humans. T3 concentrations in Sprague-Dawley rats also showed statistically significant gender differences, but not in any other species/strains or other analytes.
• Thyroid hormone fluctuations were observed during the 24hr time course analysis, as wells as on different days, in the dog. All hormone levels in the dog were stabilized 6 hours after food administration, so we recommend collecting blood samples from dogs after 6 hours post food administration or after fasting.
• The rT3/T3 ratios were relatively consistent across preclinical species and humans except in monkey, which was higher than others; the diagnostic value of this ratio will be further investigated by comparing thyroid diseased samples in toxicology studies with the generated reference ranges above.
References & Acknowledgements:
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