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Anal. Chem. 1982, 5 4 , 1188-1190
Conversion of Urinary Selenium to Selenium( IV) by Wet Oxidation Morteza Janghorbani"
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Nuclear Reactor Laboratory and Laboratory for Human Nutrition and Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
Blll T. G. Tlng Nuclear Reactor Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
Ara Nahapetlan and Vernon R. Young Laboratory for Human Nutrition and Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Ouantltatlve converslon of trlmelhylselenonlum chloride and rat urlne Se labeled biologically with '%e to Se(1V) was studled using the oxidant mlxtures HNO, HC10, (+HCI) and H202(+HCI). The results showed that neither form HNO, of selenlum Is quantltetlvely converted to Se( I V ) with HNO, H202(+HCI) but the mlxture HNO, HCIO, (+HCI) brings about satlsfactory conversion. On the basis of these results, a scheme was developed for converslon of urlne Se to Se(IV). This scheme could then be followed wlth fluorometric, neutron actlvatlon, or any other Se measurement scheme whlch operates on Se( I V ) as the measured entlty.
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Excretion of selenium in urine constitutes the major pathway for homeostatic regulation of body selenium under normal conditions of intake (1,2). The chemical forms of selenium in urine have not yet been completely characterized, but a major excretory metabolite appears to be trimethylselenonium [Se(CH&+] species (2-5). In rats, this metabolite may account for 20-50% of total urine selenium, while the only human study available suggests that 21 70 of the radioactivity present in urine between 5 and 10.5 h following intravenous injection of a dose of 75Se0,2-was excreted in this form (2). In addition, other but as yet uncharacterized forms may constitute a significant portion of urine selenium ( 2 , 3 ) . For instance, Burk (2) suggests that about 34% of the urine radioactivity is present in a fraction termed U-2. Of the potentially applicable methods for selenium analysis, the fluorometric method of Watkinson (6) or a suitable modification (7) appears to be the most widely used. The method is based on the reaction of a chelating agent (2,3diaminonaphthalene, DAN) with Se(1V) and subsequent measurement of its fluorescence a t 520 nm. Although this approach has been extensively used for measurement of selenium in various animal and plant tissues, it does not appear to have been validated for selenium analysis in urine. For an accurate measure of total urine selenium, all native forms of the element must be converted completely, or at least reproducibly, to Se(N). This is accomplished by wet oxidation of the sample with a variety of oxidant mixtures, most commonly HC104 + " O B (6, 71, "OB + HzOz (81, "OB + H@04 (9),or a suitable combination thereof ( I O ) . Satisfactory use of these oxidant mixtures is contingent upon three POtentially troublesome assumptions: (1)complete conversion 'To whom correspondence should be addressed at the Nuclear Reactor Laboratory.
of native forms of selenium to selenite-selenate, (2) reconversion of any selenate formed to selenite, and (3) prevention of significant loss of selenium during the wet oxidation steps. Several investigators have shown that loss of selenium could be prevented in open wet oxidation systems if charring of samples is avoided (6,10-14),but this may be difficult when HZSO, is a component of the mixture. Conversion of selenate to selenite is achieved by boiling the wet ashed sample in 6 N HC1 for a suitable length of time (7,13,14).However, the first assumption, or the complete or reproducible conversion of native forms of selenium to selenite-selenate has not been validated and unless this is achieved accurate analysis of urine selenium could not be obtained. This problem has been recognized by some investigators who have found that part of urinary selenium resists digestion (2). Olson et al. (7) have also referred to this problem but in relation to the loss of Se during wet ashing rather than to its incomplete conversion to selenite-selenate. Because there has not been a systematic investigation of the analysis of selenium in urine, we have explored this problem and present our results in this paper.
EXPERIMENTAL SECTION A series of chemical and animal studies were carried out to investigate various aspects of this problem and the most relevant experiments are briefly outlined below. (1) Chemical synthesis of Se(CH3)3fCl-.Trimethylselenonium chloride was synthesized and purified in the authors' laboratories according to the published method of Palmer et al. (4). Selenium analysis of the purified compound showed a selenium gravimetric factor of 0.505 as compared with the theoretical value of 0.495. (2) Animal labeling studies. Adult male Sprague-Dawley rats (CharlesRiver Breeding Laboratories,Wilmington, MA) were each intubated with single doses of either Na25Se03, [75Se]selenomethionine, or [7SSe]selenocystine.In each case, 20 pCi of 75Se was given supplying 600 pg of total selenium. Urine was collected and pooled from all treatments for the following 48 h to provide suitable size We-labeled sample for analysis. The details of these studies will be given in a separate communication. (2-20 mCi/mg Se), [75Se]selenomethionine(0.6-4 mCi/mmol), [7sSe]selenocystine(25-250 mCi/mmol), and 75Se042(2-20 mCi/mg Se), purchased from Amersham Corp. (Arlington Heights, IL) were used without further purification. All other chemicals used were analytical reagent grade and used as purchased. (4) Instrumentation. Quantitative recoveries of radiotracer, V e , were based on measurements of the recovered radioactivity as APCD-precipitate on a double-layer of filter paper, and its comparison with radiotracer standards prepared by dropwise addition of a suitable amount of radiotracer solution on identical double-layer of filter paper and its subsequent drying. Care was taken when preparing the standards to reproduce the size of the APCD precipitate from the samples. The counting instrumen-
0003-2700/82/0354-1188$0l.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
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Table I. Precipitation Behavior of APCD in Relation to Different Forms of Selenium form of' selenium % recovery in precipitatea 75Se03275Se10,275Se(CH,),+
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5.5 i. 0.4 0.8 i. 0.4
a Data are mean i:1 standard deviation of five replications; a suitable dose of tracer was added to a small volume of distilled water. In the case of 75Se0,2-5 mL of concentrated HNO, + 20 mL of concentratted HCl were added and solution was boiled to ensure complete reduction of radiotracer to selenite form. In each case, 0.2 mg of Fe3+was used as carrier and precipitation was carried out at pH 2.0 i: 0.1 with 5% (w/v) aqueous APCD solution. Adjustment of pH was made with H,SO,",OH. The recovered radioactivity was compared with standards prepared by pipetting accurate aliquots of original tracer o n similar filter paper and allowing to dry.
tation used was a Canberra 8180 multichannel analysis system equipped with a high-resolution(2.0 kev), high-efficiency (nominal 15%) Ge(Li) detector (Canberra Industries, Meriden, CN), with suitable y-shield. Sample position was reproduced carefully by insertion of the sample fiiter paper between specially constructed plastic holders which could be reproducibly inserted in the detector sample holder.
RESULTS AND DISCUSSION (A) Precipitation Behavior with APCD. In order to study conversion of different forms of selenium to Se(IV), we employed APCD (ammonium pyrrolidindithiocarbamate) as the precipitant for Se(1V) (15).This chelating agent has been used extensively for precipitation of various elements, including Fe and Zn (16). The data given in Table I were obtained to show the precipitation behavior of this agent a t pH 2.0 f 0.1 with respect to 75Se02-,75Se042-,and 75Se(CH3)3+ (Me,Se). The partial recovery of Se02- in the precipitate may, in fact, be due either to the presence of small amounts of Se032-in the tracer solution or to a reduction of the Se04'during the test procedure. On the basis of these results, we have concluded that this agent is suitable as indicator of selenium conversion to selenite and employed APCD precipitation as a means of determining the extent of conversion of MeaSe or S e 0 1 ~to~ Se032-. (B) Reduction of Se042-to Se032-. Following wet oxidation of the organic samples, the published procedures recommend boiling the resultant solution with concentrated HC1 in order to reconvert any Se042-formed back to Se032(13, 14). We tested the completeness of this conversion by the following procedure: 0.05 mL of the tracer solution of Na275Se04(Se content 3.5 ng) was boiled with 5 mL of concentrated HNO, + 25 mL of concentrated HC1 for 30 min. Following cooling, solution pH was adjusted to 2.0 f 0.1 with H2S04-NH40H, 0.2 mg of Fe3+ carrier added, and precipntation carried out with 5% (w/v) aqueous APCD solution. The recovery in the precipitate of the radiotracer for five replications was 109 f 4%. We have found that reduction of Se02- to S e 0 2 - does not take place unless HNO, is present. The reasons for this observation are not clear but might be related to perhaps catalytic action of HNO, in the electron transfer step. This observation may not be of much practical consequence to urine analysis since the solution resulting from wet oxidation of an organic sample will usually contain some HNO,. We have found out that 30 min is sufficient, under our experimental conditions, to provide reproducible conditions for this reaction but have not studied this parameter extensively. (C) Kinetics of MesSe Conversion. A SOX-125 (Glas-Col Apparatus Co., Terre Haute, IN) heating mantle holding six 125-mL flat-bottom flasks, each fitted with an air condenser,
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I2 I6 $0 24 28 32 316 40 24 48 2'2 26 6 0 T i m e , minutes
Figure 1. Kinetics of conversion of Me,Se (A) and rat urine 75Se(6) Se(1V)-Se(V1)with HNO, HCIO., (+HCI).
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was employed to study the time dependence of conversion of selenium from either MeaSe or 75Se-labeledrat urine to Se(1V)-Se(V1). To each flask was added either Me3Se (containing 936 pg of Se) or 1mL of 75Se-labeledrat urine, followed by addition of 10 mL of HC104 (60%) and 10 mL of concentrated HN03. The samples were heated and timing was begun as soon a3 white fumes of perchloric acid appeared. At intervals, an appropriate sample was removed from the heater and allowed to cool. Following this decomposition step, 20 mL of concentrated €IC1was added to each flask, the solutions were boiled for about 30 min, cooled, and pH adjusted to 2. f 0.1, and the APCD precipitation was carried out (see footnote in Table I for details). The results of this experiment are given in Figure 1. This experiment was also carried out with Me3Se corresponding to about 10 pg of Se and essentially similar kinetic results have been obtained. These data illustrate the importance of sufficient decomposition time in regard to complete conversion of MeaSe to Se(1V)-Se(V1). In addition, the kinetics of this conversion for 75Se-labeledrat urine are similar, albeit small apparent differences, to those for genuine Me,Se. The observed differences could be related to possible inconsistencies in our timing method or could be real and related to the fact that not all of the 7!'Se in rat urine is in the form of Me3Se; a significant portion it3 possibly present as the yet uncharacterized U-2 fraction ( 2 ) . We have studied potential volatilization of MeaSe during decomposition and find no significant loss. These findings indicate that the observed underrecoveries reported by Olson et al. (7, 13) are probably related to incomplete conversion of urinary selenium to Se(1V)-Se(V1) during the decomposition step rather an any loss of selenium via volatilization. Of course, with the fluorometric method of analysis either mechanism would result in underestimation of selenium, but incomplete conversion would not be apparent from the radioactive recovery studies conducted by Olson et al. ( 7 , 1 3 ) . Thus, we believe that the correct interpretation of the observations made by Olson et al. is that there was an incomplete conversion of urine Se to Se(1V)-Se(VI), and not due to loss of Se through volatilization. We have repeated these studies using HNO, H202as the oxidant mixture and have found that use of this mixture also leads to incomplete conversion to Se(1V)-Se(V1). In a typical experiment with this mixture, about 10% of radioactivity was recovered in the APCD precipitate with the remainder present in the supernatant. (D) Reproducibility of Me3Se Conversion. Employing 30 min as the optimum decomposition time, we have studied the reproducibility of 75Se-labeled rat urine conversion to Se(1V)-Se(V1). The experimental procedure used was as indicated under section C above with 1 mL of 75Se-labeled urine plus 25 mL of unlabeled human urine as the sample. The results from five replications showed radiotracer recovery
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
in the precipitate of 105 f 2%. The slight overrecoveries arise from unavoidable counting geometric differences between the radioactivity standard and the APCD precipitates as the standards were prepared by simple deposition of 1 mL of 75Se-labeledrat urine on top of a double layer of filter paper and subsequent drying, while the samples were in the form of APCD precipitate on similar size filter paper. The results are consistent with complete conversion of urine Se to Se(IV)-Se(VI) and its subsequent complete conversion to Se(1V) and quantitative precipitation with APCD. (E) Scheme for Wet Oxidation of Urine. As a result of these studies, we have developed the following scheme for complete conversion of urine Se to Se(1V) which could then be measured fluorometrically with the existing methodology (6, 7) or with neutron activation analysis following APCD precipitation as described by us previously (8, 16): To a 125-mL flat-bottom flask add 25 mL of urine sample, 5 mL of concentrated HC104,and 20 mL of concentrated HN03 plus two glass beads. Boil the solution on Glas-Col series sox-125 heating mantle with air condenser until about 20 min after solution becomes colorless. After cooling, add 20 mL of concentrated HC1, boil for 30 min, and let cool. Filter any precipitate that may be present. We have tested the reproducibility of this scheme for human urine (unlabeled) as follows: six 25-mL aliquots of fresh human urine were subjected to the procedure given above. Subsequently selenium from each solution was precipitated with APCD at pH 2.0 f 0.1 (see footnote of Table I for details), the resulting precipitate was redissolved in hot HN03 and the precipitation step repeated. This procedure is necessary to remove as much Na and C1 as possible which would interfere with the subsequent steps in the method. The final precipitate was redissolved in a small amount of hot concentrated HN03, transferred to irradiation vials, dried and subjected to neutron activation analysis employing the (n,r) 77mSe(H = 17.6 s) reaction as described previously ( 4 1 6 ) . The results of six replicate measurements indicated a coefficient of variation (percent relative standard deviation) of 7.3%. The major portion of the variance associated with these measurements is related to the expected measurement uncertainties of the neutron activation step as discussed previously (8). Although these results do not indicate explicitly the completeness of conversion of human urine Se to Se(1V) before the neutron activation step, the observed reproducibility coupled with experimental results obtained from 76Se-labeledrat urine and MeaSe recoveries strongly suggest that complete conversion of human urine Se to Se(1V) does take place. In our earlier reported work (8) we have employed H N 0 3 HzOzas the oxidant mixture. The completeness of con-
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version of Se to Se(1V) had been established for various tissues as well as urine based on spiking with 75Se032-since biologically labeled materials were not available at that time. We had observed a coefficient of variation of 15-17% for replicate measurements on human urine using either 74Se(n,r) 75Seor (n,r) 77mSereaction, and this was higher than the values of 10% or less observed for human blood, feces, or NBS SRM 1577 (bovine liver). We had suggested a potential lack of homogeneity for these somewhat larger values for urine analyses as compared to tissues or our expected precision of neutron activation analysis. It now appears that these observations are really related to lack of complete conversion of urine Se to Se(IV), the extent depending on the fraction of urine Se originally present as Me3Se, or other chemical forms that might resist conversion with H N 0 3 HzOz. In light of the importance of the nature of the oxidant mixture used during wet oxidation of urine samples, the reluctance associated with use of HC104 as a component of oxidant mixtures, and the possibility of existence of multiple chemical forms of selenium in various other biological materials, we are now systematically evaluating the nature of oxidant mixtures in relation to complete conversion of selenium to Se(1V) in various tissues and will report our findings as they become available.
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LITERATURE CITED (1) Underwood, E. J. "Trace Elements in Human and Animal Nutrition", 4th ed.; Academic Press: New York, 1977. (2) Burk, R. F. I n "Trace Elements in Human Health and Disease"; Prasad, A. S., Ed.; Academic Press: New York, 1976;Voi 11. (3) Kiker, K. W.; Burk, R. F. A m . J . Physiol. 1974, 227, 643-646. (4) Palmer, I.S.;Gunsaius, R. P.; Haiverson, A. W.; Olson, 0. E. Blochim. Biophys. Acta 1970, 208, 260-266. (5) Byard, J. L. Arch. Biochem Biophys 1969, 130, 556-560. (6) Watkinson, J. H. Anal. Chem. 1966, 3 8 , 92-97. (7) Olson, 0.E.; Palmer, I.S.; Cary, E. E. J . Assoc. Off. Anal. Chem. 1975, 58, 117-121. (8) Janghorbani, M.; Ting, B. T. G.; Young, V. R. Am. J . Clin. Nutr. 1981, 3 4 , 2816-2830. (9) Christian, G. D.; Knoblock, E. C.; Purdy, W. C. J . Assoc. Agric. Chem. 1965, 48, 877-884. (IO) Neve, J.; Hanoeg, M.; Moiie, L. Mikrochim. Acta 1980, I , 259-269. (11) Grant, A. B. N . Z . J . Scl. 1963, 6 , 577-588. (12) Gorsuch, T. T. Ana/yst(London) 1959, 8 4 , 135-173. (13) Olson, 0.E.; Palmer, I.S . ; Whitehead, E. J. I n "Selenium in Biochemical Analysis"; Giick, D., Ed.; Wiiey: New York, 1973. (14) Watkinson, J. H. I n "Selenium in Biomedicine"; Muth, 0. A,, Oidfieid, J. E., Weswig, P. H., Eds.; AVI Publishing Co.: Westport. CT. 1967. (15) Stary, J. "The Solvent Extraction of Metal Chelates"; Pergamon Press: New York. 1964. (16) Ting, B. T. G.; Janghorbani, M.; Young, V. R. Modern Trends in Activation Analysis, Toronto, Canada, June 1981.
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RECEIVED for review January 11, 1982. Accepted March 18, 1982. This work was supported in part by a grant from the National Institutes of Health (1-ROI-Ca-27917-01).
