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Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

ABSTRACT: Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17β-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17β-estradiol were highly correlated with R² of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17β-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.

SUBMITTER: Nouri MZ

PROVIDER: S-EPMC7447131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 ?L) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of (13)C12-T4 internal standard in plasma extractions was: 99?±?1.8 % in rainbow trout, 85?±?11 % in fathead minnow, 73?±?5.0 % in mummichog, 73?±?1.7 % in sockeye salmon, and 80?±?8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.

Project description:The Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5-triiodo-L-thyronine-thyronine (T3), 3,3´,5´-triiodo-L-thyronine-thyronine (rT3), 3,3´-diiodo-L-thyronine (3,3´-T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2-14.02% and 0.4-17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6-20.7% and 2.5-15.6% respectively, and accuracies were 80.2-128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´-T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency.

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Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS.

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