HPLC separation and analysis method to evaluate molecules involved in the arginine-ornithine cycle in Phytomonas

L. FRACCAROLI; P. TORRES; M. RUIZ; M. MARCORA; V. DE PINO; C. CARRILLO

Congreso; XXIX Reunión de la Sociedad Argentina de Protozoología; 2017

Phytomonas spp. are trypanosomatid parasites that infect a variety of plants and can cause diseases that affect regional economies, mainly in South America. Little is known about Phytomonas biology; however, recently published genomic data provide new venues for research. We are interested in the study of arginine ? ornithine cycle with special focus on the enzymes involved in the conversion of arginine-citrulline-ornithine.To confirm our previous evidences about the presence of NOS and ODC and the absence of ADC and AGMATINASE enzymes in this cycle, we set up a method for separation and analysis of products and intermediates using high-performance liquid chromatography (HPLC) that involves precolumn derivatization with o-phthaldialdehyde, C18 analytical column and fluorescence detection. We testedtwo mobile phase solutions (MP): 1- MPA: 0.1M sodium acetate, pH7.2/MPB: 100% methanol and 2- MPA: 25mM phosphate buffer pH7.5/MPB: acetonitrile:methanol:water 45:45:10, both at three different temperatures (15, 25 and 40ºC). Under these different conditions we separated a mixture of standard aminoacids: arginine, ornithine, citrulline, agmatine, lysine, glycine and threonine. We found that mobile phase 2 at 40ºC was the best condition to obtain individual peaks for each aminoacid with a detection limit of at least1uM. As a first in vivo approach, Phytomonas Jma was cultured in a semi-defined media (SDM-79) and extracts obtained from 2 and 10x106 cells were deproteinized and neutralized. The HPLC chromatogram, under the selected conditions, showed the seven aminoacid peaks well resolved and a low background signal. Furtheranalysis will be made to analyze the aminoacid profile under different culture conditions. In conclusion, we have set up a HPLC protocol that will be useful to analyze Phytomonas metabolism. This method could also be used to analyze aminoacids in a variety of biological samples such as physiological fluids, tissue extracts and different cell types.