Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography

doi:10.1016/0378-4347(92)80093-6

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 574, Issue 1, 7 February 1992, Pages 17-21

https://doi.org/10.1016/0378-4347(92)80093-6 Get rights and content

Abstract

A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described. Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde. The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media. The limit of detection is 20–30 pmol, and the response of the fluorescence detection is linear up to 4 nmol. The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation. The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine [N-(2-aminooxyethyl)-1, 4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane] in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.

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Citation Excerpt :
We homogenized PBMCs in buffer containing 25 mmol/L Tris/HCl (pH 7.4), 0.1 mmol/L EDTA, and 1 mmol/L DTT. Polyamine concentrations in the homogenates were measured with high-performance liquid chromatography [12]. Supernatant fractions of centrifuged homogenates were used to perform ODC and SSAT activity assays as described previously by Jänne and Williams-Ashman [13] and Bernacki et al. [14], respectively.
The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N¹-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N¹-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression–induced myeloproliferative disease in mice.
Ethanol-induced impairment of polyamine homeostasis - A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome
2014, Biochemical and Biophysical Research Communications
Citation Excerpt :
Tissue samples from the placenta, yolk sac, embryo heads and embryo trunk were frozen in liquid nitrogen and stored at −70 °C for polyamine measurements. The natural polyamines (spermidine, spermine and putrescine) were measured by high-performance liquid chromatography according to the method of Hyvönen et al. [15]. Briefly, the samples were homogenized in 300 μl of ice-cold buffer (25 mM Tris pH 7.4, 0.1 mM EDTA, 1 mM dithiothreitol), 100 μl aliquots of the homogenates were precipitated with 100 μl of 10% sulphosalicylic acid containing 20 μM diaminoheptane and the supernatant fractions were taken for HPLC analysis after centrifugation.
Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting.
CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined.
No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction.
Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.
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Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N¹-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT – acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress.
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Journal of Chromatography B: Biomedical Sciences and Applications

Abstract

References (17)

Differentiation

J. Biol. Chem.

Biochem. J.

Ann. Rev. Biochem.

Cancer Res.

Biochem. Biophys. Res. Commun.

Biochem. J.

Cited by (120)

Spermidine/spermine N<sup>1</sup>-acetyltransferase activity associates with white blood cell count in myeloid leukemias

Ethanol-induced impairment of polyamine homeostasis - A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome

Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine- N<sup>1</sup>-acetyltransferase in human hepatoma HUH7 cells

Acetylated derivatives of C-methylated analogues of spermidine: synthesis and interaction with N<sup>1</sup>-acetylpolyamine oxidase

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells

Protective Effect of Resveratrol against Testicular Dysfunction Induced by Forced Swimming Exercise in Adult Rats