Acta Chim. Slov. 2003, 50, 185-198. 185 SELENIUM DETERMINATION IN WHOLE BLOOD, PLASMA AND SELENOPROTEIN P BY HYDRIDE GENERATION ATOMIC FLUORESCENCE SPECTROMETRYf Darja Mazej, Ingrid Falnoga, Vekoslava Stibilj Jožef Štefan Institute, Ljubljana, Slovenia f This paper is dedicated to Dr. Marjan Dermelj, on the occasion ofhis70thbirthday. Received 28-01-2003 Abstract Selenium was determined in whole blood, plasma and plasma protein fractions SeP and (GSH-Px+Alb) obtained by affinity chromatography. For the digestion of whole blood and plasma samples a simple digestion procedure using H2S04, H202 and V205 in H2S04 solution was developed, while for the decomposition of plasma protein fractions HN03 and H202 were sufficient. After both procedures detection of Se was made by HG-AFS and this combination gave reliable results. Dilution of the GSH-Px+Alb fraction was reduced by lyophilisation of pooled subfractions. The main advantages of the described procedures are low detection limit (0.2 ng Se/g of solution) and the large number of samples analysed in a short tirne as only two analyses are needed for determination of the percentage of Se in SeP in plasma sample. The procedures developed were used to determine selenium in real samples from which an average mass fraction of selenium in whole blood of Slovenia residents of 87 ± 13 ng/g (n=43) and Se in SeP of 70 ± 9% (n=5) were found. Both values are comparable with literature data for the general population. Introduction The essential trace element selenium is of fundamental importance to human health. As a constituent of selenoproteins, selenium has an enzymatic role, best-known as an antioxidant scavenging free radicals, and as a catalyst for the production of active thyroid hormone. About 35 selenoproteins have been identified, though for many of them their role has not been fully explained. In mammalian plasma, selenium is mainly incorporated in three proteins, selenoprotein P (SeP), extracellular glutathione peroxidase (GSH-Px) and albumin (Alb). SeP is the major selenoprotein in plasma. Ali selenoproteins so far reported contain one atom of Se as selenocysteine per molecule except SeP, which contains ten atoms of Se as selenocysteine per molecule. The function of SeP is currently unknown, although several indirect lines of evidence suggest that SeP is a free radical scavenger or Se transporter. D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma and selenoprotein P... 186 Acta Chim. Slov. 2003, 50, 185-198. Estimation of human selenium status has become more and more important. Most frequently the measurement of selenium in whole blood and its fractions and GSH-Px activity have been used as a measure of Se status. In recent times the distribution betvveen plasma proteins has become an important indicator and SeP as the major plasma protein has become an interesting marker of Se status. In this regard several separation procedures based on chromatography have been developed to separate SeP from GSH-Px and albumin. Affmity chromatography based on specific binding of SeP on sepharose-bound heparin has been most often used.4'5 Deagen et al first described a method for the separation of three major selenium containing proteins in plasma using two small columns of heparin-Sepharose and reactive blue 2-Sepharose linked together in tandem. The first column was used to remove SeP, while albumin was retained on blue-Sepharose. GSH-Px passed through both columns. Selenium was determined spectrofluorimetrically.4 Harrison et al made some changes to this procedure. The same columns were used independently of each other and smaller subfractions were collected to decrease the dilution effect of serial columns and joint fractions. Because a different detection system was used, electrothermal atomic absorption (ET-AAS), the type of buffer was changed from phosphate to acetate. Se determinations by ET-AAS were faster but less sensitive.5 Several detection techniques have been used for the determination of the total concentration of selenium in human plasma and whole blood, including neutron activation analysis (NAA), molecular fluorescence spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), isotope dilution mass spectrometry, ET-AAS, hydride generation atomic absorption spectrometry (HG-AAS) and hydride generation atomic fluorescence spectrometry (HG-AFS).6 Various mixtures of nitric, perchloric and sulphuric acids with hydrogen peroxide in open or closed systems are often used for 7 R digestion of blood samples. ' For determination of selenium in protein fractions after affinity chromatography where the concentrations of selenium are very low, the amount of fraction available is limited and the number of fractions is numerous, the choice of detector is narrower, mostly limited to ET-AAS and HG-AFS. For HG-AFS detection systems the digestion of samples is necessary. The aim of this work was to develop and optimize a very simple, reliable and sensitive method for the determination of selenium in whole blood, plasma and plasma D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma andselenoprotein P... Acta Chim. Slov. 2003, 50, 185-198. 187 protein fractions obtained after chromatographic separation. For the isolation of SeP from plasma, a published5 separation procedure was used and partly modified regarding collection and preparation of the chromatographic fractions. Harrison et al determined Se in fractions directly by ET-AAS,5 but we used digestion of fractions with nitric acid and hydrogen peroxide. The digestion procedure using sulfuric acid, hydrogen peroxide and V2O5 in H2SO4 for whole blood and plasma samples, which has already been published,9 was also modified. Both digestion procedures were followed by HG-AFS detection. Experimental Blood and plasma samples 43 blood samples (2x7 mL) from residents of Slovenia were collected at the Clinical Centre, Ljubljana in Vacutainer tubes (Becton Dickinson) containing either no anticoagulant or Na- or Li- heparin. After cooling at room temperature (20 min), the sample aliquots were centrifuged (10 minutes, 2500 rpm). The resulting plasma samples were transferred to glass tubes with covers (Labco). One part of the plasma was stored at 4 °C for not more than two days before separation. Blood and the other part of the plasma samples were stored at -20 °C prior to Se determination. Isolation of SeP from plasma by affinity chromatography a) Reagents: In chromatographic separation ultra-pure water (Milli Q, Millipore, USA) and the following chemicals were used: NaCl (Merck, suprapur), CH3COONH4 (Merck, practopur), 90% CH3COOH (Merck, suprapur), heparin (Krka, 5000 U/mL), 65% HN03 (Merck, suprapur). Equilibrium buffer (0.02 mol CH3COONH4/L, pH 6.8): 1.54 g CH3COONH4 was dissolved in 1 L of Milli-Q water. Elution buffer (0.02 mol CH3COONH4/L with 500 U/L heparin, pH 6.8): 5 mL heparin (5000 U/L) was diluted with 50 mL of equilibrium buffer. Regeneration buffers (0.02 mol CH3COONH4/L with 0.5 mol/L NaCl, pH 5 and 10): 2.92 g NaCl was dissolved in 100 mL of equilibrium buffer, and the pH was adjusted with 5% acetic acid or 5% ammonium hydroxide. b) Procedure: Proteins were separated in a glass column (30 cm x 1 cm, Econo, Bio-Rad) filled with 2 g of heparin-Sepharose CL-6B (Pharmacia). The flow rate through the column was 2.5 mL/min and was adjusted at the peristaltic pump (Ismatec, MCP 380) by D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma andselenoprotein P... 188 Acta Chim. Slov. 2003, 50, 185-198. the choice of tubing size (Tygon, LFL). The column was equilibrated with 50 mL of equilibrium buffer. 2 g of plasma were diluted with equilibrium buffer (ratio 1:1) and applied to the column. GSH-Px + Alb (fraction A) was eluted from the column with 40 mL of equilibrium buffer. SeP (fraction B) bound to heparin-sepharose was then eluted with 50 mL of elution buffer and collected in 20 subfractions, 2.5 mL each. The column was regenerated with regeneration buffers, 16 mL of each and washed with equilibrium buffer (3x8 mL) before re-equilibration. Fraction A was lyophilised to 1/5 of its volume (24 hours, -52 °C, 0.030 mbar, freeze drier LOC-1, CHRIST) and then diluted to 10 g with Milli-Q water. 200 \xL of 65% HNO3 was added to subfractions of fraction B and fraction A. Fraction A and B were stored at -20 °C before selenium determination. Selenium determination a) Reagents: For preparation of solutions and sample treatment ultra-pure water (Milli Q, Millipore, USA) and the following chemicals were used: 96% H2SO4 (Merck, suprapur), 65%) HNO3 (Merck, suprapur), 30% HC1 (Merck, suprapur), 30% H202 (Merck, p.a.), NaOH (Merck, p.a.), NaBLL (Fluka, purum p.a.), V2O5 (Merck, p.a.), Na2SeC>3 (Sigma, 98%>), selenomethioneine (SeMet, Sigma), selenocystine (SeCyst, Sigma). Solution of V2O5 in H2SO4: 3.4 g V2O5 was added to 30 mL of Milli-Q water and slowly mixed with 170 mL of concentrated H2SO4 at 4 °C. The solution was kept in darkness. 1.2% NaBFL in 0.1 mol/L NaOH: 6 g NaBH4 and 2 g NaOH were dissolved in 500 mL of Milli-Q water, filtered through a 0.45 \xm filter and stored at 4 °C before use. The solution was prepared daily. SeMet: 0.2412 mg Se/g stock solution of SeMet was prepared by dissolving 0.0598 g of SeMet in Milli-Q water. SeCyst: 0.1985 mg Se/g stock solution of SeCyst was prepared by dissolving 0.0210 g of SeCyst in Milli-Q water. Se(IV): stock solution was prepared by dissolving 0.2294 g Na2Se03 in 100.00 g 0.1 mol/L HC1 and stored at 4 °C. The mass fractions of Se in stock solutions were checked by k0-INAA.10 Solutions with lower mass fractions of Se were prepared daily by dilution of stock solution. D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma andselenoprotein P... Acta Chim. Slov. 2003, 50, 185-198. 189 b) Sample pretreatment of blood/plasma samples: 0.15 g of sample (blood, plasma) was weighed into a teflon tube (50 mL, Savillex). 1 mL 96% H2S04 and 1 mL 30% H202 were added and the tube was heated in an aluminium block at 115 °C for 15 min. After cooling 2 mL 30%> H2O2 was added and the tube was heated for 75 min at 115 °C. The solution was then cooled to room temperature, 100 \xL solution of V2O5 in H2SO4 was added and well mixed. Heating for 20 min at 115 °C followed and the solution became blue coloured. To reduce selenium to Se(IV) which was necessary for hydride generation, 2 mL of concentrated HC1 was added to the solution and heated for 10 min at 100 °C. Finally, the solution was diluted to 15 g with Milli-Q water and selenium was measured by the HG-AFS system. In the same way and with the same amount of chemicals as used for samples, a process blank was run every tirne. In the čase of standard addition, a solution of Se(IV) was added to samples before digestion. Calibration solutions prepared in the same acid media as samples, samples and blank were measured at least tvvice. It is important to emphasize that the entire process of sample treatment from weighing to measuring was made in the same Teflon tube. c) Sample pretreatment ofprotein fractions: 1 g of protein fraction was weighed into a teflon tube (25 mL, Savillex), 0.5 mL concentrated HNO3 was added and the closed tube was heated for 10 min on a hot plate. Then 0.5 mL of 30%> H2O2 was added three times and evaporated every tirne to lA volume. Reduction of selenium was made by the addition of 0.5 mL concentrated HC1 and heating for 10 min on a hot plate. The solution was fmally diluted to 2.5 g and measured by the HG-AFS system. In the same way and with the same amount of chemicals as for samples, a process blank was run every tirne. Calibration solutions prepared in the same acid media as samples, samples and blank were measured at least tvvice. And as was mentioned before it is important that the entire process of sample treatment from weighing to measuring was made in the same Teflon tube. d) Measurement by HG-AFS: The continuous flow approach of an HG-AFS system was used to merge samples with reagents. The flow of carrier (0.05 mol/L HC1, 1 mL/min) or a sample solution was mixed in a PEEK (polyetheretherketone) cross connector with HC1 (2 mol/L, 8 mL/min) and NaBIL; (1.2%(m/V) in O.lmol/L NaOH, 3 mL/min). Flow rates were adjusted at the peristaltic pump (Ismatec, MCP 380) by the choice of tubing D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma andselenoprotein P... 190 Acta Chim. Slov. 2003, 50, 185-198. size (Tygon, LFL). The hydrogen selenide (H2Se) and hydrogen which resulted from mixing in the cross piece were separated from liquid in a gas-liquid separator, swept from it with argon (260 mL/min) and dried in a Permapure dryer (Perma Pure Products). The dryer gas was nitrogen (3 L/min). H^Se was atomized in an hydrogen/air flame in an atomic fluorescence spectrometer (model Excalibur, PS Analytical) with a Photron Se Super lamp. Results and discussion Determination o/Se in blood/plasma samples Optimisation of measurement by HG-AFS was first made and involved several variables. The ranges studied and optimum values found are shown in Table 1. Table 1. Optimisation of HG-AFS system. Variable Range studied Optimal value Argon flow rate (mL/min) 100-375 260 Nitrogen fiow rate (L/min) 2-3 3 Mass fraction of NaBH4 (% m/V) 1.2 - 1.8 1.2 Cone. of HC1 for hydride generation (mol/L) 2-4 2 Cone. of HC1 in carrier (mol/L) 0 - 0.5 0.05 For Se determination in blood samples the procedure published by Tiran et al9 was slightly modified. We altered the mass of sample, temperature of digestion, tirne of reduetion of Se(VI) to Se (IV) and dilution mass. The mass of sample should be around 150 mg. At lower masses homogeneity can be a problem, while at higher masses we obtained too low and irreproducible results because of incomplete digestion, since there was not enough oxidant in solution. But if more sulphuric acid was added, problems with the density of liquid flow in continuous HG-AFS inereased. The efficiency of digestion was improved by inereasing the temperature from 100 to 115 °C. The tirne of reduetion was shortened from 20 to 10 min since experiments with radiotracer 75Se showed that reduetion is complete in this tirne.11 Because of the great sensitivity of the deteetion system used (HG-AFS), the mass of solution to which the sample was diluted, was inereased from 10 to 15 g. In this way the influence of sulphuric acid decreased. D. Mazej, I. Falnoga, V. Stibilj: Selenium determination in whole blood, plasma andselenoprotein P... Acta Chim. Slov. 2003, 50, 185-198. 191 As it is known that in hydride generation several interferences, including H2SO4 and HNO3 can be present, the standard addition technique and the reagent matched calibration curve were used. A comparison betvveen the results obtained by these two approaches was made. The results in Table 2 shows only slight differences. According to the t-test (texp