MAGHEMITE PARTICLES FOR SPERMIDINE SEPARATION

We report the optimal conditions for the separation of spermidine from different types of samples, for example blood or cancer cells and its subsequent determination by ion-exchange liquid chromatography (IEC) with UV-VIS detector. Here in, we synthesized paramagnetic particles able to isolate and immobilize spermidine from blood of patients with cancer or cancer cells and thus preconcentrate it for analysis. Dowex surface was covered by nanomaghemite (γ-Fe 2 O 3 ) or maghemite particles surface was modified by chitosan and sulfoxyethyl cellulose. The best separation properties showed Dowex microparticles. The paramagnetic particles can be used in the future for isolation of spermidine from real samples and diagnosis of cancer.


INTRODUCTION
Spermidine is a polyamine, usually isolated from sperm, located in the living cells, tissues and ribosomes [1]. Spermidine plays important role in various biological processes, such as regulation of plant growth, assisting in vitro process of transcribing RNA, and/or inhibition of nitric oxide synthase (NOS) [2]. Spermidine and spermine, are promising biomarkers of Parkinson disease (PD). Each polyamine enhance longevity via autophagy induction, under physiological conditions demonstrated ability of their metabolite to act as an diagnostic, age-related and severity of Parkinson disease [3]. On the other hand, increased levels of polyamines are toxic to cells, and can facilitate cell death based on the oxidative stress [4]. Polyamines, such as spermidine, spermine and putrescine have antioxidant properties, play major roles in the prevention of chronic diseases for example cardiovascular diseases and in the differentiation and development of immune system [5]. Currently, different methods are developed for spermidine detection. Most of them are based on chromatography (GC, HPLC) [6,7] with tandem of mass spectrometry (MS) [8], IEC [9] or CE [10]. In our study, we decided to use ion exchange chromatography with post-column ninhydrin derivatization and VIS detector for spermidine determination [10].

Chemicals
Spermidine of purity 99 % was obtained from Sigma Aldrich (St. Louis, Missouri, USA). Solution of spermidine for preparing of calibration curve was prepared in the dilution buffer Na: TDG (N3Na -0.10 g, NaCl -11.5 g, C6H8O7 -14 g per 1L H2O

Ionex chromatography
An AAA 400 (Ingos, Czech Republic) liquid chromatography apparatus was used for determination of amino acids. The system consists of a glassy filling chromatographic column and steel precolumn, two chromatographic pumps for transport of elution buffers and derivatization reagent, cooled carousel for 25 test tubes of 1.5 -2.0 mL volume, dosing valve, heat reactor, VIS detector and cooled chamber for derivatization reagent. Chromatographic columns for transfer of elution buffers and derivatization reagent are able to work at flow 0.01-10 mL•min −1 under a maximum pressure of 40 MPa. Volume of injected sample was 100 µL with an accuracy of application RSD of about 1%. A two-channel VIS detector with a 5 µL flow volume cuvette was operated at wavelengths of 440 and 570 nm.

MAN 1
Iron chloride(III) hexahydrate (2g) was dissolved in water (160 mL) and a solution of NaBH4 (0.4 g) in 3.5 % NH3 (20 mL) was added with stirring. After hydrogen evolution, the mixture was heated to boiling for 2 h and left overnight. Nanomaghemite was separated on magnet and washed several times with water. Sulfoxyethyl cellulose (0.5 g) was poured into nanomaghemite suspension, agitated overnight, separated on external magnet and dried at 40 ºC.

MAN 16
Nanomaghemite was prepared as described for MAN 1. Dowex (1 g) was poured to nanomaghemite suspension instead of cellulose.

MAN 25
In the preparation chitosan solution (1 ml, 1 %) was added with stirring to the suspension of nanomaghemite. Product was treated as in previous cases.

RESULTS AND DISCUSSION
Goal of our paper was primarily isolation and separation of spermidine, based on the adsorption of analyte on the paramagnetic microparticles and subsequent determination using ion-exchange liquid chromatography (IELC). We modified nanomaghemite with sulfoxyethyl cellulose (MAN 1), DOWEX (MAN 16) and chitosan (MAN 25). SEM characterization of the prepared microparticles is visualized of Figures 1 and 2. Paramagnetic particles on the surface of Dowex sphere are well seen on Figure 1A (MAN 16). The fibres of sulfoxyethyl cellulose covered with magnetic particles are visualized on Figure 1B and C for MAN 1, whereas the structure of MAN 15 is seen on Figure 2 at three different magnifications. All the three kinds of microparticles are stable in solution and are perfectly separated from the solution by external magnetic field. In our experiments of amines binding to paramagnetic particles, we have tested six amines. The results can be seen on graphs in Figure 3. These PMPs showed excellent properties for binding of spermidine (recovery 37.25 %) for MAN 16, 15.6 % for MAN 25 and 14.32 % for MAN1 ( Figure 3D). We used Britton-Robinson buffer with pH 2 which causes spermidine protonation that leads to positive charging of molecules due to its pI = 5.3 for spermidine. Interaction between surface of magnetic microparticles and positively charged molecules provides the binding between them. These interactions depend on isoelectric points of polyamines, which are in this mode behaving as the ion-exchangers.

CONCLUSION
In our study, we synthesized new paramagnetic microparticles able to bind spermidine that can be considered as promising biomarker of Parkinson disease. The paramagnetic microparticles (MAN 16) have potential to better isolation from the samples of plasma, cells and/or or tissue and in future can serve for application as a biosensor.