Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a lower limit of quantitation (LLOQ) of ≈3pM in induced sputum (174pg/mL) and saliva (198pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically (15)N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC-MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.

A LC-MS-MS method to detect recombinant bovine somatotropin misuse in buffalos.

Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the fair trade and the law prescriptions. For this reason, efficient and reliable analytical techniques are required to contrast rbST misuse. A few LC-MS-MS methods have been developed to detect, in cow serum, methyonil-rbST, one of the two main rbST forms available on the market. The other form, which is widespread, is identical to the most abundant variant of bovine somatotropin (bST) and differs from the buffalo somatotropin for one amino acid in the N-terminus. For this reason, it is technically possible to distinguish both rbST forms in serum of buffalos. In this work, we describe a novel LC-MS-MS-based method, capable to quantify, with a high sensitivity and selectivity, the methyonil-rbST and the other bST-identical recombinant form in buffalo serum, previously purified using a solid-phase extraction procedure. The method was internally validated and used to analyse 152 serum samples, collected from eight buffalos administered with rbST for a period of 3 months, according to conventional protocols. The obtained results confirmed the suitability of the method in the detection of illegal hormonal treatments. Graphical abstract ᅟ.

Highly sensitive antibody-free µLC-MS/MS quantification of rhTRAIL in serum.

BACKGROUND: We describe an antibody-free approach to quantify rhTRAIL(WT) (wild-type) and its closely related death receptor 4 selective variant rhTRAIL(4C7) in human and murine serum by multiplex LC-MS/MS on a microfluidics interface. METHODOLOGY: Enrichment of rhTRAIL was performed by strong cation-exchange (SCX) followed by immobilized metal affinity (IMAC) solid-phase extraction. This was followed by trypsin digestion and using methionine-containing signature peptides after fully oxidizing the methionine residue with 0.25% (w/w) hydrogen peroxide. CONCLUSION: Absolute quantification was reaching down to 0.5 ng/ml for rhTRAIL(WT) (8.5 pM) and 2 ng/ml for rhTRAIL(4C7) (34 pM) in 100 µl human serum. To support preclinical studies in mice, the analysis was optimized further, for a sample volume of 20 µl murine serum.

Effect of Hypothyroidism and Hyperthyroidism on Tissue Thyroid Hormone Concentrations in Rat.

BACKGROUND AND OBJECTIVE: The present study was aimed at determining the effects of experimental hypothyroidism and hyperthyroidism on tissue thyroid hormones by a mass spectrometry-based technique. METHODS: Rats were subjected to propylthiouracil treatment or administration of exogenous triiodothyronine (T3) or thyroxine (T4). Tissue T3 and T4 were measured by liquid chromatography tandem mass spectrometry in the heart, liver, kidney, visceral and subcutaneous adipose tissue, and brain. RESULTS: Baseline tissue T3 and T4 concentrations ranged from 0.2 to 20 pmol ∙ g(-1) and from 3 to 125 pmol ∙ g(-1), respectively, with the highest values in the liver and kidney, and the lowest values in the adipose tissue. The T3/T4 ratio (expressed as a percentage) was in the 7-20% range in all tissues except the brain, where it averaged 75%. In hypothyroidism, tissue T3 was more severely reduced than serum free T3, averaging 1-6% of the baseline versus 30% of the baseline. The extent of tissue T3 reduction, expressed as percentage of the baseline, was not homogeneous (p < 0.001), with liver = kidney > brain > heart > adipose tissue. The tissue T3/T4 ratio significantly increased in all organs except the kidney, averaging 330% in the brain and 50-90% in the other tissues. By contrast, exogenous T3 and T4 administration produced similar increases in serum free T3 and in tissue T3, and the relative changes were not significantly different between different tissues. CONCLUSIONS: While the response to increased thyroid hormones availability was similar in all tissues, decreased thyroid hormone availability induced compensatory responses, leading to a significant mismatch between changes in serum and in specific tissues.

Modulation of gene expression by 3-iodothyronamine: genetic evidence for a lipolytic pattern.

3-Iodothyronamine (T1AM) is an endogenous biogenic amine, structurally related to thyroid hormone, which is regarded as a novel chemical messenger. The molecular mechanisms underlying T1AM effects are not known, but it is possible to envisage changes in gene expression, since delayed and long-lasting phenotypic effects have been reported, particularly with regard to the modulation of lipid metabolism and body weight. To test this hypothesis we analysed gene expression profiles in adipose tissue and liver of eight rats chronically treated with T1AM (10 mg/Kg twice a day for five days) as compared with eight untreated rats. In vivo T1AM administration produced significant transcriptional effects, since 378 genes were differentially expressed in adipose tissue, and 114 in liver. The reported changes in gene expression are expected to stimulate lipolysis and beta-oxidation, while inhibiting adipogenesis. T1AM also influenced the expression of several genes linked to lipoprotein metabolism suggesting that it may play an important role in the regulation of cholesterol homeostasis. No effect on the expression of genes linked to toxicity was observed. The assay of tissue T1AM showed that in treated animals its endogenous concentration increased by about one order of magnitude, without significant changes in tissue thyroid hormone concentration. Therefore, the effects that we observed might have physiological or pathophysiological importance. Our results provide the basis for the reported effectiveness of T1AM as a lipolytic agent and gain importance in view of a possible clinical use of T1AM in obesity and/or dyslipidaemia.

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid, an endogenous end product of thyroid hormone metabolism.

BACKGROUND AND PURPOSE: 3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects. EXPERIMENTAL APPROACH: TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 µg·kg(-1) ) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H1 receptor antagonist pyrilamine (10 mg·kg(-1) ) or the H2 receptor antagonist zolantidine (5 mg·kg(-1) ). TA1 (1.32 and 4 µg·kg(-1) ) was also given i.c.v. to mice lacking histidine decarboxylase (HDC(-/-) ) and the corresponding WT strain. KEY RESULTS: TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 µg·kg(-1) ), stimulation of learning (1.32 and 4 µg·kg(-1) ), hyperalgesia (0.4, 1.32 and 4 µg·kg(-1) ) and hyperglycaemia (1.32 and 4 µg·kg(-1) ). All these effects were modulated by pyrilamine and zolantidine. In HDC(-/-) mice, TA1 (1.32 and 4 µg·kg(-1) ) did not increase plasma glucose or induce hyperalgesia. CONCLUSIONS AND IMPLICATIONS: Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.