High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives

doi:10.1016/S0378-4347(00)84307-8

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 221, Issue 2, 12 December 1980, Pages 227-235

https://doi.org/10.1016/S0378-4347(00)84307-8 Get rights and content

Abstract

The separation of the natural polyamines and their monoacetyl derivatives by high-performance reversed-phase liquid chromatography is reported. Octane sulfonate was used to form ion pairs with the polycations and the o-phthalaldehyde method for post-column derivatization. The method allows polyamine and acetylspermidine determinations directly from tissue extracts and body fluids without pre-purification.

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DFMO was kindly provided by Hoechst Marion Roussel, Cincinnati, OH, USA and dissolved in phosphate buffered saline (PBS). The polyamines were determined in cellular homogenate by HPLC and normalized to total protein as described by Seiler and Knodgen [11]. Cellular polyamine concentration was expressed as nmol/mg total protein.
Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re)stenosis.
The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd.3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression.
DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd.3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury.
Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process.
Phospho-sulindac (OXT-328), a novel sulindac derivative, is safe and effective in colon cancer prevention in mice
2010, Gastroenterology
Citation Excerpt :
Cyclooxygenase (COX) activity and prostaglandin E2 (PGE2) levels were determined by immunoassays, following the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). Cells treated with test compound(s) for 24 hours were assayed for polyamine levels by high-performance liquid chromatography (internal standard: 1,7-diaminoheptane).9 For the spermidine/spermine-N1-acetyltransferase (SAT1) activity, cells (3 × 106) treated with test compound(s) were harvested by scraping, disrupted by sonication in buffer [10 mmol/L Tris-HCl (pH 7.5), 2.5 mmol/L DTT, 1 mmol/L EDTA], and pelleted.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective cancer chemopreventive agents. However, chronic administration of NSAIDs is associated with significant side effects, mainly of the gastrointestinal tract. Given these limitations, we synthesized phospho-sulindac (P-S; OXT-328), a novel sulindac derivative.
Here, we evaluated the safety and efficacy of P-S in preclinical models, including its mechanism of action with human colon cancer cell (HCCC) lines and animal tumor models.
(1) Compared with sulindac, P-S is much more potent in inhibiting the growth of cultured HCCCs and more efficacious in preventing the growth of HT-29 xenografts in nude mice. P-S also prevents the growth of intestinal tumors in Apc/Min mice. (2) In combination with difluoromethylornithine (DFMO), P-S reduced tumor multiplicity in Apc/Min mice by 90%. (3) P-S is much safer than sulindac as evidenced by its in vitro toxicologic evaluation and animal toxicity studies. Mechanistically, P-S increases the intracellular levels of reactive oxygen and nitrogen species, which are key early mediators of its chemopreventive effect. Moreover, P-S induces spermidine/spermine N¹-acetyltransferase enzymatic activity, and together with DFMO it reduces polyamine levels in vitro and in vivo.
P-S displays considerable safety and efficacy, two pharmacologic properties that are essential for a potential cancer chemopreventive agent, and thus merits further evaluation.
Identification and characterization of a diamine exporter in colon epithelial cells
2008, Journal of Biological Chemistry
SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N¹-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.
Polyamine acetylation modulates polyamine metabolic flux, a prelude to broader metabolic consequences
2008, Journal of Biological Chemistry
Citation Excerpt :
The supernatant extract was assayed for polyamines by reverse phase HPLC. We obtained sharp polyamine peaks with good separation (Fig. 3) by introducing major modifications to a previously described procedure (36) as detailed below. Polyamines in 50 μl of each acid extract were injected onto an autosampler (model 717 Plus, Waters Associates, Milford, MA) using a C-18 column (250 × 3 mm; 5 μm) (Alltech Associates, Deerfield, IL) with an acetonitrile/sodium acetate gradient buffer system and detected with an o-phthalaldehyde postcolumn derivatization system.
Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N¹-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 μm 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by ∼5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.
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High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives

Abstract

J. Chromatogr.

J. Pharm. Sci.

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr.

Methods Enzymol.

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Anal. Biochem.