Abstract:The objective of the present study was to investigate the uptake and speciation changes of Se enriched leek by sodium-Selenite, sodium- selenate and barium-selenate in a green house experiment. The sandy loam soil was chosen with Se concentration—ppm. Se concentration applied was 160µg Se/Kg, 1,2 and 3 mg Se/Kg in each treatment was studied during time period of 3 months . For total Se determination samples were digested by 2.5 mL HNO3(65%) and 2.5 mL H2O2(–%) was used and the obtained Se concentrations ranged between —- and —- in Se enriched leek. Speciation was done by using enzymatic extraction (protease) on reversed phase Altima C8 column and PRP-X-Anion exchange column to evaluate the potential speciation changes on Se-enriched leek. Majority of Species indentified was Selenite, Selenate, Seleno-methyl-Seleno Cystine,Seleno methionine,Se-cystine. In Sodium selenate 42.75% and in barium selenate treatment 39.17% remained inorganic selenate where as Sodium selenite in sodium selenite fertilized 26.76% remained as inorganic Se. On the basis of these investigations it is concluded that Selenite is a better source than selenate to form better organic species which were described previously as anti carcinogenic and unlike other allium species leek also has ability to accumulate Se.

Soil type pH Org. C(g kg_1),N(g kg_1),P(mg kg_1),K(mg kg_1),Mg(mg kg_1),Ca(mg kg_1),Na(mg kg_1),Se(mg kg_1)INTRODUCTION

Selenium is an essential trace element for humans and animals. The most important source of selenium is diet. Selenium deficiency can cause different diseases [2], mainly in those regions where selenium level in soil is noticeably low [3]. Beside the total content of selenium, the chemical form in which selenium is present is also most important due to the differences in bioavailability and toxicity of the different forms as organic form of certain Se species showed anti carcinogenic activity [7,8]. Plants that are capable of accumulating higher amounts of selenium during cultivation and transforming it to an appropriate chemical form are one of the potential sources for enhancement of Se daily intake. The Se concentration and the species formed is depend on the selenium form available in the soil. Several studies described allium family species(chives, onions, garlic) have capability to accumulate higher selenium concentration and transformed the soil inorganic form into organic form in the plant. But there is not much attention towards leek which also belongs to allium family has chosen in this study.

Leek is one of the major commercial crop in Belgium, the official estimation leek cultivated was 4200 ha of which 1000-1500 ha was cultivated for the industry. The production of leek per ha was between 25 and 60 ton/ha with a mean production value 35 ton/ha as per PCG, vegetable experimental station, Belgium. The current study was to investigate the potential Se accumulation and speciation forms of leek plants on different Se forms in green house experiments.

2. Experimental

2.1. Instrumentation

An ICP-MS PerkinElmer (Sunnyvale, CA, USA)was applied for total Se and speciation analysis as an element-specific detector. The ICP-MS was fitted with a Babington nebulizer, a

Scott double pass spray chamber and a Peltier cooling system was used for elemental and species determination. The ICP-MS coupled with a liquid chromatographic system (HPLC-ICP-MS) from PerkinElmer (Sunnyvale, CA, USA) was used. It consisted of a P680 HPLC pump, an ASI-100 automated sample injector and an Elan DRC-e ICP-MS detector (PerkinElmer, Sunnyvale, CA, USA). A Hamilton PRP-X100 anion exchange column from Grace, Belgium and Altima C8 column (250 mm – 4.6 mm I.D., 5 μm, 120 Å) from Agilent, Unitedkingdom were used as stationary phase both equipped with a guard column with the

same stationary phase material. HPLC-ICP-MS conditions are presented in Table1.

Selenium species extraction was carried out by using a shaker fitted incubator chamber(——) and The obtained extracts were centrifuged on an Eppendorf centrifuge 5804 F34-6-38. Total Se determination was carried out by using microwave digestion apparatus from Mars (——)For mixing Se in the soil thoroughly in an homogenized way a mixer from (—–) was used.

2.2. Reagents and standards

Seleno compounds, namely, sodium selenite (Na2SeO4), sodiumselenate( Na2SeO3), barium selenate (BaSeO4),selenomethionine (SeMet),seleno cystine (Secys), Se-methyl-l selenocysteine (MeSeCys) were purchased from Aldrich (St. Louis, MO, USA) and Gamma-glutamyl-Se-methyl-selenocysteine(γ-glut-cyst) , Gamma-glutamyl-Se-methyl-seleno methionine(γ-glut-meth) were purchased from pharmaSe (—–).For chromatographic purposes, citric acid obtained from(—),Hepta fluorobutryic acid obtained from (—-) anfd to adjust desired pH ammonium hydroxide was obtained from (—-).For the sample preparation procedures, protease XIV was purchased from Sigma, while concentrated HNO3 from(—-) and H2O2 from (—) were purchased from Merck. MilliQ® (MQ) water from Water Systems Ltd., High Wycombe UK) was used throughout the whole experiment. The chromatographic standard and other working solutions were prepared daily as required.

Samples

Cultivation and preparation of Se-enriched green onions and chives.

For this study, commercially available Leek (Allium fistulosum) plantlets of poulton variety were purchased. The boxes were filled with 30 kilograms of soil in each box and the soil was completely mixed in mixing rotator with the required concentration of Se. In each box 8 plants were planted in two rows and were watered with deionized water. Three types of Se forms Na2SeO4, Na2SeO3 and BaSeO4 with 4 different concentrations of 160µg Se/Kg, 1,2 and 3 mg Se/Kg in each box seperately.To prevent pests pestiscide(——-) was sparyed twice over a period of 3 months.

After 3 months, plants were removed from boxes and washed first with tapwater to remove the soil traces and then washed with deionised water.Then the weight of each plant was recorded and cut the entire plant into pieces manually and tranferred into polyethylene boxes and shock frozen them immediately with liquid nitrogen and stored at -80°C and then freeze dried by liophiliser and powdered with milling apparatus.

3.1. Extraction

3.1. Total Se determination

Sample preparation for total Se determination. For the determination of the total Se content of leek samples open destruction method was optimized, 0.2 g sample was placed into centrifuge tube and dissolved in a mixture of 2.5 ml concentrated HNO3 and 2.5 ml of 30% H2O2. After 16 h of contact time, total digestion was carried out at in a microwave (ref) to obtain a totally digested sample. Final solutions were then made up to 50.0 ml in a volumetric flask with deionized water. For the validation of the system, a certified reference material of plant origin was applied namely, BCR-CRM-402 (white clover), whose certified total Se content is 6.7+/-0.27 mg/g. For total Se determination quality control of the system was done by three replicates of BCR-CRM-402 with each sample batch. The obtained samples total Se were further determined by the method of standard addition and external standard calibration.

Enzyme hydrolysis: 80 mg of the enzyme Protease XIV and 0.2 g sample dissolved in 5 mL of water and added to in a 10 mL centrifuge tube and the mixture was shaken for 24 h at 37 °C. After extraction the mixture was centrifuged for 30 min at 14,000 rpm/min (Eppendorf 5804R). The supernatant was separated from sediment and filtered through a 0.22 µm Millex GV filter. Supernatants and sediments were stored at -20°C until analyses for total Se and Se speciation were carried out.

4.Results and Discussion

4.1Soil results:

The sandy-loamy soil used in this study was analyzed for total elemental concentrations(ref), pH, conductivity, mineral nitrogen and organic carbon( tab-no).

4.2 Total Se determination

The total Se concentrations in plants were shown high Se concentrations present in the plants fertilized with Na2SeO4 in all the four concentrations applied which is 10 folds higher than Na2SeO3 and BaSeO4.Among the three types of fertilization Na2SeO3 has low total Se compared with other forms in all the four concentrations. The results were shown in table[1]

4.3 Determination of Se species

Both inorganic or organic Se compounds can be present in plants as they may incorporate into high molecular weight compounds such as proteins. Terry et al. [7] suggested a Se pathway in higher plants. The Se uptake from soil by roots was metabolized by the plant to form organoselenium compounds is an approach as the organic Se species showed ant carcinogenic properties, which are either accumulated or volatilized. In the current study, presence of selenospecies in leek plants was confirmed by anion exchange and reversed phase chromatography. An existing method for the chromatographic separation of selenoamino acids by isocratic elution anion-exchange HPLC [30] was used for the separation of selenium species contained in leek. The resulting HPLC conditions (see Table 1 for details) provided good selectivity for the seven selenoamino acids, as illustrated in Fig. 2a. For confirmative qualitative analysis, a separate chromatographic method based on reversed-phase HPLC [24] (see Table 1 for details) was used to determine the seven Se species, as shown in Fig. 1 made possible the retention and separation of seven selenoamino acids, as shown in Fig. 2. The two inorganic Se species was poorly resolved in RP-HPLC was quantified in anion-exchange. The

[7] N. Terry, A.M. Zayed, M.P.D. Souza, A.S. Tarun, Annu. Rev. Plant Physiol.

Plant Mol. Biol. 51 (2000) 401-432

Article name: Determination Of SE Concentration And Speciation Changes Biology essay, research paper, dissertation

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