T-3

A fingernail clipping is a promising specimen for evaluating the medium-to-long term status of low-molecular weight hormones. However, the nail concentrations of thyroid hormones and their clinical significance remain poorly understood. In this study, a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for simultaneously quantifying triiodothyronine (T₃) and thyroxine (T₄) in the thumbnail clippings. The nail T₃ and T₄ were extracted with 50 % ethanol and purified using a Strata-X cartridge, then injected into the LC/ESI-MS/MS. ¹³C₆-T₄ was used as the internal standard for both T₃ and T₄. Validation results demonstrated that the method was precise (relative standard deviations in five repetitive measurements were ≤ 4.5 %) and accurate (analytical recovery rates were 98.3-103.3 %). The lower limits of quantification were 0.20 ng/g for both thyroid hormones. By using this method, we found that the nail concentrations of T₃ (0.74 ± 0.26 ng/g, mean ± standard deviation) were at the same level as those of T₄ (0.90 ± 0.27 ng/g) in euthyroid subjects (n = 40). This result might be caused due to the greater free and albumin-bound fractions of circulating T₃ compared to T₄. The patients with Graves' disease showed significantly higher nail T₃ and T₄ concentrations than the euthyroid subjects. The patients with hypothyroidism due to Hashimoto's thyroiditis tended to have lower nail T₃ and T₄ concentrations compared to the euthyroid subjects. Thus, the developed method would be useful for evaluating the medium-to-long term status of thyroid hormone production and has sufficient clinical applicability.

C-type natriuretic peptide (CNP) is a multifaceted paracrine factor that regulates vital physiological functions of the cardiovascular system. The present study was designed to investigate the anti-hypertrophic activity of CNP in the presence and absence of HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA) in T₃-induced H9c2 cells. The H9c2 cells were treated with T₃ (10 nM) for 48 hours to induce hypertrophic growth. T₃ alone-treated cells exhibited an increase in cell size (p < 0.001) as compared with control H9c2 cells. CNP treatment effectively reverted the increased cell size by 61 %, and also altered the expression of hypertrophic marker genes. Interestingly, a significantly enhanced anti-hypertrophic activity was noticed in CNP co-treatment with SAHA, and also a more significant decrease in the expression of hypertrophic marker genes (α-sk, and α-MHC) was also observed. Moreover, CNP treatment with SAHA effectively reversed the T₃-induced changes in the expression of Npr1 and Npr2 genes in H9c2 cells. In addition, CNP co-treated with SAHA reversed the T₃-induced abnormal calcium influx by normalizing of CaMKII and SERCA2a. The findings of the present study clearly show that CNP co-treatment with SAHA significantly enhances the CNP-mediated anti-hypertrophic activity, probably by restoring Npr1 and Npr2 gene expression and calcium influx. Taken together, we conclude that CNP in combination with SAHA represents a novel therapeutic approach for the treatment and management of cardiac hypertrophy and heart failure.