Abstract
Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.
Highlights
Polyamines are aliphatic amines with low molecular weights that are involved in the regulation of many cellular functions, including translation, transcription, signal transduction, cell proliferation, cell differentiation, apoptosis, and cell stress responses [1]
Chloroform, sodium bicarbonate, sodium carbonate, ammonium acetate, trifluoroacetic acid, perchloric acid, sodium hydroxide, acetone, and dansyl chloride were obtained from Sigma-Aldrich, whereas ethyl acetate was obtained from Merck
The acquisition of dansylated analytes and their stable-isotope-labeled internal standards was performed in positive electrospray ionization mode, and the source conditions were optimized to obtain the maximum response for the [M + H]+
Summary
Polyamines are aliphatic amines with low molecular weights that are involved in the regulation of many cellular functions, including translation, transcription, signal transduction, cell proliferation, cell differentiation, apoptosis, and cell stress responses [1]. The major polyamines in mammalian cells are spermidine (SPD), spermine (SPM), and their precursor, diamine putrescine (PUT). They are present in a variety of biological matrices in both their natural and acetylated forms. In addition to polyamines and acetylpolyamines, the polyamine metabolome includes their precursor amino acids (i.e., arginine (ARG), ornithine (ORN), and lysine (LYS)) as well as the polyamine-related metabolites produced by nonmammalian organisms, such as cadaverine (CAD), agmatine (AGM), and 1,3-diaminopropane (1,3-DAP) [4]. Urinary N1, N12-diacetylspermine (N1,N12-DiAcSPM), has been proposed as a biomarker for the diagnosis and prognosis of different types of cancer [3,5,6,7,8], and the plasma levels of N-acetylputrescine (N-AcPUT) and 1,3-DAP have been proposed as biomarkers for the early diagnosis of lung cancer [9]
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