Separation of N1- and N8-acetylspermidine isomers by reversed-phase column liquid chromatography after derivatization with dansyl chloride

Stefanelli, Claudio; Carati, Daniela; Rossoni, Carmen

doi:10.1016/s0378-4347(00)83690-7

Cited by 48 publications

(27 citation statements)

References 11 publications

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“…Aliquots of 20 μl were subjected to Western blotting analysis. To measure the polyamine content in the extracts, 10 μl of sample was diluted in 0.3 ml 0.3 M perchloric acid and analyzed by HPLC as described [21], except that a linear gradient from 35 to 75% of solvent B over 40 min was applied. The mean concentration of endogenous polyamines in S‐3 extracts was approximately 10 μM putrescine, 20 μM spermidine and 30 μM spermine.…”

Section: Methodsmentioning

confidence: 99%

Spermine triggers the activation of caspase‐3 in a cell‐free model of apoptosis

Stefanelli¹,

Bonavita²,

Stanic³

et al. 1999

FEBS Letters

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Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post-nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro-apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.z 1999 Federation of European Biochemical Societies.

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Section: Methodsmentioning

confidence: 99%

Spermine triggers the activation of caspase‐3 in a cell‐free model of apoptosis

Stefanelli¹,

Bonavita²,

Stanic³

et al. 1999

FEBS Letters

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“…PA were determined by the method of Stefanelli et al [1986]. Briefly, the cells were centrifuged and homogenized in 4 vol 0.3 M perchloric acid.…”

Section: Polyamine Assaymentioning

confidence: 99%

Changes in polyamines, c‐myc and c‐fos gene expression in osteoblast‐like cells exposed to pulsed electromagnetic fields

Mattei

Gagliano

Moscheni

et al. 2005

Bioelectromagnetics

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Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.

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“…and Davies, 1977;Niitsu et al, 1993) or liquid chromatography (LC) with fluorescence (Cichy et al, 1993;van Eijk et al, 1996;Kai et al, 1979;Price et al, 1987;Redmond and Tseng, 1979;Seiler and Knödgen, 1985;Stefanelli et al, 1986;Weiss et al, 1997) or mass spectrometric (Feistner, 1994) detection have been described.…”

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confidence: 99%

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer‐forming fluorescence derivatization using 4‐(1‐pyrene)butanoyl chloride

Yoshida

Harada

Nakano

et al. 2004

Biomedical Chromatography

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A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.

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Separation of N1- and N8-acetylspermidine isomers by reversed-phase column liquid chromatography after derivatization with dansyl chloride

Cited by 48 publications

References 11 publications

Spermine triggers the activation of caspase‐3 in a cell‐free model of apoptosis

Spermine triggers the activation of caspase‐3 in a cell‐free model of apoptosis

Changes in polyamines, c‐myc and c‐fos gene expression in osteoblast‐like cells exposed to pulsed electromagnetic fields

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer‐forming fluorescence derivatization using 4‐(1‐pyrene)butanoyl chloride

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