Anal. Chem. l 9 W S60,2734-2737
2734
Determination of Selenium Metabolites in Biological Fluids Using Instrumental and Molecular Neutron Activation Analysis Alan J. Blotcky Medical Research, Veterans Administration Medical Center, Omaha, Nebraska 68105 Alireza Ebrahim a n d Edward P.Rack* Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
Optimized procedures have been developed for the determlnatlon of total seienlum, trlmethylselenonium(TMSe) Ion, and selenite (SeOt-) ion In urlne and serum and for total selenoamlno acids In urine by anion exchange chromatography and molecular neutron actlvatlon analysis. For urlne samples containing greater than 40 ng of Se/mL, determinaion can be performed. For urine tion for TMSe and Se0;samples containing nondetectableSeOt- ion whose concentratlon dlfferentlai between the total selenium and TMSe is 140 ng of Se/mL, total selenoamlno aclds can be detennlned by derlvatlzatlon and anion exchange chromatography. For urlne samples whose differential between total selenium and TMSe SeOc- is greater than 30 ng of Se/mL, a preclpltatlon/copreclpitatlon technique employing Ba( NO,), and (NH4),SO4 must be performed prior to the subsequent steps. The procedures were evaluated for over 100 specknens from normal and diseased subjects.
+
Selenium is a vital micronutrient with a narrow range of intake consistent with health; outside of this narrow range deficiency disease and toxicity occur (1-7). While there have been various techniques (8-11) for the determination of total selenium in biological matrices such as urine (0.019-0.184 pg of Se/mL), serum (0.055-0.180 pg of Se/mL), liver (0.180-0.660 pg of Se/g), and bone (1.0-8.95 pg of Se/g) (8, 12), only a few procedures have been published for the determination of individual selenium metabolites in biological fluids (13-15). These results are necessary for describing the biological pathways for selenium metabolism in living organisms. Recently we reported two studies (16,17) for the determination of selenite and TMSe in urine by ion exchange chromatography and molecular neutron activation analysis. In our routine analyses for total selenium, TMSe, and selenite ion for normal and disease state urine, we found significant unaccounted contributions to the total selenium. This is in agreement with earlier studies (13,18, 19) that reported unidentified selenium metabolites. Since selenoamino acids such as selenomethionine, selenocystine, and selenocysteine have been hypothesized (20, 21) as metabolites in the biological pathways of selenium incorporation and excretion in living systems, we report procedures for determination of total selenoamino acids in urine. In addition we optimize the experimental procedures for the determination of selenium metabolites in urine and serum by instrumental and chemical neutron activation analysis. EXPERIMENTAL SECTION Reagents and Solvents. ACS reagent grade lithium hydroxide (anhydrous) 99.3% from Alfa Products (Danvers, MA), concentrated ammonium hydroxide from J. T. Baker (Phillipsburg,NJ), ammonium sulfate from EM Science (Cherry Hill, NJ), and formaldehyde solution (37.9%) and barium nitrate from Mal0003-2700/88/0360-2734$01 SO/O
linckrodt (Paris, KY) were used without further purification. Selenium(1V) oxide, Puratomic (99.999%) from Johnson Mathey (Seabrook, NH) was used as the selenium standard. Sodium selenite, seleno-DL-cystine,and seleno-DL-methionine were obtained from Sigma Chemical Co. (St. Louis, MO). Dimethyl selenide, obtained from Columbia Organic Chemical Co., Inc., (Camden,SC), was used to prepare trimethylselenonium according to the procedure described by Palmer et al. (15). Fluoraldehyde (o-phthalaldehyde/2-mercaptoethanol)reagent solution from Pierce (Rockford, IL) was used to derivatize the amino acids. Water used in all phases of this work was treated by passing through a Culligan (Northbrook, IL) S-series reverse osmosis system. Preparation of the Resin and Columns. Several chromatographic resins and columns from Bio-Rad Laboratories (Richmond, CA) were evaluated for use in this method. Analytical anion exchange resins such as Bio-Rad AG-1-X8 in acetate form (100-200 mesh) and Bio-Rad AG-2-X8 in chloride and nitrate forms (200-400 mesh) along with borosilicate Econo-Columns in various sizes (0.5 X 20 cm, 0.7 X 20 cm, and 0.7 X 10 cm) were evaluated. A combination of AG-2-X8 resin in the nitrate form and a 0.7 X 10 cm borosilicate column resulted in a rapid technique for analysis of total selenoamino acids in urine. The anion exchange columns for analysis of selenoamino acids were prepared by slurry loading 0.7 X 10 cm columns with AG2-X8 resin in the nitrate form. For analysis of TMSe and SeO2ions, 0.7 X 20 cm columns were packed with a slurry of AG-2-X8 resin in the chloride form. Prior to each analysis, 20 mL of 0.5 M LiOH solution was passed through each column to equilibrate the resin. Neutron Irradiation. Clear polystyrene 5-mL sample tubes and polyethylene caps from Sarstedt, Inc. (Princeton, NJ), were used for all samples. Samples were irradiated for 20 s at a thermal neutron flux of 3.1 X lo1’ n cm-2 5-l in the Omaha Veterans Administration Medical Center TRIGA nuclear reactor by means of a pneumatic transfer tube. Radioassay. All irradiated samples were allowed to decay for 20 s and counted for a live time of 20 s in a 58-cm3closed-end coaxial well Ge detector with a relative efficiency for 6oco of 10.4%. A well detector was used since it was shown to be 5.5 times as efficient as a 70-cm3solid Ge(Li) detector for the 162 keV 77mSe y and exhibited less geometry dependence. The analyst must evaluate the sample volume radioassayed since the increased count rate associated with a large volume will result in an increase in elapsed real time when counting in the live-time mode of the multichannel analyzer. Because of the short half-life of 77mSe(tl/z = 17.4 s), the counting rate diminishes at too rapid a rate for an accurate correction of decay and counting time (22). For example, volumes of 1 mL or less in an urine sample gave assay values of 0.10 pg of Se/mL. For the same urine sample, volumes of 2.0 and 3.0 assayed at 0.06 and 0.04 pg of Se/mL, respectively. y spectra analyses were accomplished by using a Nuclear Data (Schaumburg, IL) ND 680 4096-channel analyzer employing ND “Peak” software. Storage and Sampling of Urine Prior to Analysis. To evaluate a method of minimizing sedimentation that occurs in urine and ensure constant sampling, urine was treated with concentrated “*OH 1+5 (v/v) and 0.5% by volume of 37% formaldehyde and allowed to stand 24 h before analyzing. There is no statistical difference between treated and untreated urines. 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
1I
Dala Reduction
ImL UrinelSwum
Multichannel Annlyzer
1 . Oelerminalion 01 TMSe and 50032C~rOmltoQraDhY 0 Anion Exchanpe
Figwe 1. Block diagram for determination of total selenoamino acids in urine with (A) nondetectable Se032-and (B) detectable Se032-.
Sampling position has no effect on the determinations. In order to preserve urine, it is routinely treated with 0.5% by volume of 37% formaldehyde solution upon receipt of the sample from the donor. The effect of ultrasound mixing was also evaluated by analyzing duplicate sets of urine samples freshly collected and those stored in a refrigerator for 7 days. Upon receipt of the urine it was fixed with formaldehyde solution and divided into four aliquotes. Two of the aliquotes were analyzed immediately, and two were stored in a refrigerator for 7 days. One of the aliquots of the fresh urine was ultrasounded for 30 min while the other was not. Triplicate samples were taken from the top, middle, and bottom of each flask. Two consecutive samplings were performed after 15-min intervals resulting in a total of 27 samples for each set. The same procedure was repeated for the two aliquotes stored for 7 days. Application of both the t test and f test indicated that there was no statistical difference between the nonultrasound and ultrasound mixed samples. Determination of Total Selenium in Urine and Serum. One-milliliter quantities of urine and serum were analyzed for total selenium content by instrumental neutron activation analysis. These analyses are essential for establishing a selenium material balance in the biological fluids. Determination of Trimethylselenonium and Selenite Ions in Serum and Urine. Our reported procedure for determination of TMSe and Se03" ions in urine, which is now applicable for serum, was previously described (17). A method of additive spikes was employed for determination of TMSe and Se032-in urine. This technique worked equally well for both these biological fluids. Determination of Total Selenoamino Acids in Urine. Total selenoamino acids in urine were determined by derivatizing the amino acids with 0-phthalaldehyde (OPA) and 2-mercaptoethanol (231,which led to retention of derivatized selenoamino acids on the anion exchange chromatographic column. Two separate procedures were developed for determination of total selenoamino acids because the selenite ion was found to interfere with the analysis. The complete outline of these two procedures is presented in Figure 1for samples with nondetectable and detectable amounts of selenite ion, respectively. The procedure for determination of selenoamino acids in urine samples with nondetectable selenite ion calls for a precolumn derivatization of amino acids with OPA followed by anion exchange chromatography. This enabled us to elute out TMSe and capture the amino acids on the resin. The resins were then irradiated and radioassayed for selenium content. For determination of selenoamino acids in urine samples with detectable selenite ion, a procedure was utilized that employed a precolumn precipitation/coprecipitation of Se032-by Ba(NO& and (NH&304solutions followed by derivatization of amino acids and anion exchange chromatography. Removal of selenite ion prior to derivatization of amino acids was necessary because this ion, upon treatment with the OPA/2-mercaptoethanol derivatizing solution, was partially retained on the resin and interfered with the determination of selenoamino acids. This procedure enabled us to precipitate selectively Se03*, elute out TMSe, and capture selenoamino acids on the resin which was later radioassayed. For each analysis, 1mL of urine was diluted with 1mL of water, -and the urine sample was alkalized to a pH of 10-11 by addition of 1mL of 0.5 M LiOH in order to dissociate selenoamino acids from protein binding sites (24).Lithium hydroxide solution rather
I '
1
I
I
1
2735
1
STOP
' I
IW Delermmalion of Total Selenoamlno Acids by Pro-Column Derlvelization and Anion Exchange ChromatoaraDhy
Figure 2. Schematic for making deckion for determlnatbn of (A) total selenium in urlne and serum, (B) total selenoamino acids in urine with nondetectable Se0:-, and (C) total selenoamino acids in urine with detectable Se032-.
than ammonium hydroxide is used for the denaturing step in the selenoamino acid analysis because ammonium ions react with the amino acid derivatizing reagent (25) and lowers the availability of OPA for amino acids present in the urine. After complete elution of the column with 20 mL of 0.5 M LiOH, the resin was allowed to dry and later radioassayed after transferring the resin to a polystyrene vial. Dry resin will minimize the contribution of lDO(Ey = 0.198 MeV) to the y-ray spectrum of the sample. RESULTS AND DISCUSSION Development of Optimum Procedures for Determination of Selenium Metabolites in Urine. Because urine is an admixture of multiple metabolites and ions whose concentration range varies over wide limits, it is difficult to propose a single procedure to compensate for all of the variables associated with each subject's urine sample. The schematic in Figure 2 describes the methods adopted for determining total selenium, TMSe, Se032-,and selenoamino acids in urine. Part A of Figure 2 was also successfully applied to serum. As shown in part A of Figure 2 only samples with total selenium concentration greater or equal to 40 ng of Se/mL were suitable for reliable TMSe and Se032-analyses because of the dilution factors involved in each analysis and the 10 ng of Se/mL detection limit. Of the samples that were used for the determination of TMSe and Se032- those that qualified for total selenoamino acid analysis had to meet the criteria specified in parts B and C of Figure 2. During the course of this study, it was found that selenite ion upon treatment with OPA derivatizing solution was retained on the resin and interfered with total selenoamino acid analysis. As can be seen in Table I the individual selenium compounds that are not treated with OPA derivatizing agent all elute from the column when they are in an aqueous matrix. Conversely, when these selenium compounds are treated with the derivatizing reagent, TMSe elutes out, Se032-is partially retained, and selenocystine and selenomethionine are held on the resin. This suggested that the Se0:- interfered with the selenoamino acid analysis and needed to be removed prior to derivatization. For samples with nondetectable Se032-, a straightforward derivatization was performed,and for samples with detectable SeOS2-a precolumn precipitation/coprecip-
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
~~
Table I. Behavior of Selenium-ContainingCompounds in Presence and Absence of OPA Derivatizing Solution in Water OPA absent % eluted
OPA present % captured
% eluted
% captured
compound
from column
elution vial
on column
from column
elution vial
on column
selenite ion trimethylselenonium ion selenomethionine selenocystine
99 97 95 98
4-6 2+3 4-12 4+5
ND" ND ND ND
22 100 ND ND
7 + 15 2+3
65 ND 100 100
" Nondetectable. Table 11. Assay of Resins Containing Selenium Metabolites in Presence of OPA Derivatizing Solution in Urine after Precipitation of Selenite Ion Prior to Derivatization
metabolite
wg of Se added to resin
wg of Se captured on resin
recovery
selenite ion trimethylselenonium ion selenocystine selenomethionine
1.527 0.182 0.436 1.322
0.582 f 0.021 ND" 0.427 f 0.011 1.258 f 0.033
38 ND 98 96
Table 111. Determination of Total Selenium in Urine of Subject (limit of detection 10 ng of Se/mL)
sample Omaha, normal subjects Omaha, cancer patientsD Louisville, disease patients'sd Louisville, kidney patientscae patient with high selenium excretion patient with high selenium excretion after chelating with germanium
total Se,O ng of Se/mL 58 52 18 32 162
39 5
251
sample
total Se,O ng of Se/mL
confidence limit (90%)
TMSe,b ng of Se/mL
SeO:-,b ng of Se/mL
1 2 3 4 5
119 141 128 172 134
20 7 12 34 19
85 36 69 132 75
ND' 15 ND 24 71
%
" Nondetectable.
confidence limit (90%)
Table IV. Determination of Total Selenium, Trimethylselenonium Ion, and Selenite Ion in Pooled Serum Samples (limit of detection 10 ng of Se/mL)
" Determined by INAA. *Determined by the method of additive spikes. 'Nondetectable. Table V. Determination of Total Selenium, TMSe, SeOt-, and Total Selenoamino Acids in the Urine Containing Greater Than 40 ppb Selenium
no. of subjects
4 4
13 13 15 11
3
1
37
1
Separate determinations were made on individual urine collections. Between 33 and 60 individual determinations were used to calculate the mean. *Patients with Hodgkins lymphoma, NonHodgkins lymphoma, leukemia, and breast cancer prior to bone marrow transplant. Samples collected at University of Louisville School of Medicine, Department of Biochemistry. Noncancer diseases such as coronary symptons, pneumonia angina, and hypertension. CPatientswith end-state renal disease and on continuous amulatow peritoneal dialysis (CAPO).
itation with Ba(N03)2and (NH4)2S04prior to derivatization was employed. In an attempt to determine if an individual constituent of the OPA derivatizing solution such as OPA, 2-mercaptoethanol, and borate buffer was causing the Se03to be held on the resin, the procedure that was outlined in part B of Figure 1was carried out on Se02- with individual constituent of OPA solution. In all cases Se032-eluted out. Table I1 shows the results of the precipitation/derivatization method for determination of selenoamino acids in urine. As can be seen the amount of Se02- that is trapped on the resin is 36% of total Se032-,which means that the precipitation/ coprecipitation of Se032- is an effective way to lower the concentration of Se03" in urine. Determination of Selenium Metabolites in Urine Employing the Optimized Procedure. By use of the optimized procedures described in the previous section, 54 urine samples from normal and disease state subjects were analyzed for total selenium and are presented in Table 111. Five different pooled serum samples were also analyzed for total selenium and the results are shown in Table IV. For each individual sample
sample Omaha, NE, normal subjects
Omaha, NE, cancer patients Hodgkinslymphoma non-Hodgkinslymphoma leukemia breast cancer Louisville, KY, disease patients Louisville, KY, kidney patients patient with high selenium patient with high selenium after chelating with germanium
total selenium, ng of Se/mL
TMSe,' ng of Se/mL
SeO:-: ng of Se/mL
total selenoamino acids,' ng of Se/mL
97 f 20 70 f 17 50 f 17 160 f 17 89 f 22 51 f 10 65 f 12 56 f 12 54 f 15 50 f 34
23 56 22 63 21 20 ND 13 ND 11
NDb 13 ND ND 41 ND 65 38 ND ND
NM' NM NM ND NM NM NM NM NM NM
69 f 8 47 f 6 48 f 22 83 f 20 73 f 18 48 f 13 50 f 20 60 i 19 82 f 31 45 f 10 56 f 18
57 3 44 ND 80 44 14 ND 6 20 ND ND 15 ND
NM NM NM NM NM NM NM NM NM LDd LD
68 f 28 160 f 29
34 27 ND 53 ND ND ND 19 57 33 29 ND 18 52
260 f 37
41
ND
300
83 f 67
64
LD 100
Selenium weight in indicated species. bND = nondetectable. NM = not measured. LD = limit of detection; lower than 40 ng (as selenoamino acids) per milliliter of urine.
the schemes described in Figure 2 were employed to make decisions as to the viability of further analysis in determining TMSe, selenite, and total selenoamino acids. These deter-
Anal. Chem. IQW, 60, 2737-2744
minations are presented in Tables IV and V. Of the 54 urine samples studied only 25 samples met the criteria for TMSe and Se02- determination, and after complete anion exchange chromatographic separation of TMSe and Se032-in these urine samples, we found that 28% of the samples had nondetedable TMSe and 48% had nondetectable Se032-. As can be seen in Tables IV and V, the selenium concentration of serum is greater than that of urine per unit volume. After determination of TMSe and Se032- in all qualified urine samples, only seven urine samples (28%) met the requirement for determination of selenoamino acids. While we did not attempt to ascertain clinical correlation among urine samples, correlations will be reported in future studies. By use of the method of Currie (26) the detection limit for determination of total selenoamino acids is 40 ng of Se/mL and for determination of total selenium, TMSe and Se032-is 10 ng of Se/mL.
ACKNOWLEDGMENT The authors thank K. P. McConnell and Margaret Tempero for supplying the urine samples and G. T. Hansen for carrying out some of the experimental work. LITERATURE CITED (1) McConnell. K. P.; Broghamer, W. L., Jr.; Blotcky, A. J.; Hart, 0. J. J . Nutr. 1975, 705, 1026-1031. (2) Broghamer, W. L., Jr.; McConnell, K. P.; Blotcky, A. J. Cancer 1978, 3 7 , 1384-1388. (3) Broghamer, W. L., Jr.; McConnell, K. P.; Blotcky, A. J. Cancer 1978, 4 7 , 1462-1466. (4) Shamberger, R. J.; Rukovena, E.;Longfeld, S. A.; Tyko, S.; Deodhar, C. E.; Wlllls, C. E. J. Natl. Cancer Inst. ( U S ) 1973, 50, 863-670. (5) Wlllett, W. C.; Polk, B. F.; Morris, J. S.; Stampfer, M. J.; Pressel. S.; Rosner, B.; Taylor. J. 0.; SchneMer, K.; Hames, C. 0.Lancet 1982, 2 , 130- 134.
2737
(6) Levander, 0. A. Fed. Roc. Fed. Am. Soc.Exp. Bbl. 1985, 4 4 , 2579-2583. (7) Buel, D. N. Semin. Oncol. 1983, 70. 311-321. (6) Iyengar. G. V.; Kollmer. W. E.; Bower, H. J. M. The Elemental Conpslfion of Human Tlssues and Bo@ Fluids ; Verleg Chemle: New York, 1978. (9) Cornelis, R.; Speecke, A.; Hoste, J. Anal. Chim. Acta 1975, 78. 317-322. (10) Versleck, J.; Cornells. R. Anal. Chim. Acta 1980, 776, 217-254. (11) Ishizak, M. Talanfa 1978. 25. 167-169. (12) Versieck, J. CRC Crlf. Rev. Clin. Lab. Sci. 1985, 22. 97-184. (13) Oyamada, N.; Ishlzaki. M. Jpn. J. Ind. Health 1983, 25, 319. (14) Nahapetian, A. T.; Young, V. R.; Janghorbani, M. Anal. Biochem. 1984, 740, 56-59. (15) Palmer, L. S.; Fischer. D. D.; Haalverson, A. W.; Olson, 0. E. Biochim. BiophyS. Acta 1980, 777, 336-342. (16) Blotcky, A. J.; Hensen, G. T.; melanio-Buencamino. L. R.; Rack, E. P. Anal. Chem. 1985, 57, 1938-1941. (17) Blotcky, A. J.; Hansen. G. T.; Bofkar, N.; Ebrahlm, A.; Rack, E. P. Anal. Chem. 1987, 5 9 . 2063-2066. (18) Byard, J. L. (Arch. Biochem. Blgphys. 1989. 730, 556-560. (19) Nahapetian, A. T.; Janghorbani, M.; Young, V. R. J. Nutr. 1883, 773. 401-411. (20) BUrk. R. F. J . NU?. 1988, 776, 1584-1586. (21) Sunde, R. A.; Hoekstra, W. G. Biochem. Bbphys. Res. Commun. 1980, 9 3 , 1181-1186. (22) Blotcky, A. J.; Arsenault, L. J.; Rack, E. P. Anal. Chem. 1973, 4 5 , 1056-1 060. (23) Roth, M. Anal. Chem. 1971, 4 3 , 880-882. (24) Kessler, G.; Pileggi, V. J. Clln. Chem. (Winston-Salem, N . C . ) 1988, 74, 811-619. (25) Goyal, S. S.; Rains, D. W.; Hauffaker, R. C. Anal. Chem. 1988, 60, 175-179. (26) Currle, L. A. Anal. Chem. 1988, 4 0 , 586-589.
RECEIVED for review May 31,1988. Accepted September 13, 1988. This research was supported by the US.Department of Energy, Division of Chemical Sciences, Fundamental Interaction Branch, under Contract DE-FG02-84ER13231.A003 and a University of Nebraska Research Council NIH Biomedical Research Support Grant No. RR-07055.
Dispersion Coefficient and Moment Analysis of Flow Injection Analysis Peaks Stephen H. Brooks,I Daniel V. Leff, Maria A. Hernandez Torres, and John G. Dorsey* Department of Chemistry, University of Florida, Gainesville, Florida 3261 1
The dlsperslon coefflclent ( D )Is the most popular peak descrlptor In flow Injectlon analysis (FIA). Yet thls concept of dlsperslon ylekls no dlred information descrlblng peak shape and no Information In the t h e domain. Using an exponentlaUy modlfled Gausslan peak shape model and previously derlved equatlons, we examine the second moment (variance) of single-Hne flow Injection peaks and use thls as a fundamental descriptor of the FIA response curves. Unllke the dlsperslon coefflclent, the second moment Is shown to obey a h e a r relatlonshlp wlth respect to flow rate and to yleld valuable lnformatlon In the presence of a chemlcal reactlon. Reportlng descriptors of an FIA response curve as a variance offers several advantages over the classlcal dlsperslon coeffIclent: peak wldth (In unlts of time or volume) Is lmmedlately obtalnable from the variance, yleldlng a more direct measure of sample throughput; varlous FIA manifolds can be readily compared from thelr varlance values, and the lndlvldual contrlbutlons to the total peak variance (Includlng the contribution of the chemlcal reaction to the total varlance) can be easlly obtalned through the addltlvlty of variances. 'Present address: IC1 Pharmaceuticals Group, Wilmington, DE 19897.
IC1 Americas,
Inc.,
0003-2700/88/0360-2737$01.50/0
Flow injection analysis (FIA) is f i i y established as a rapid, precise, efficient, and extremely versatile analytical tool. Ruzicka and Hansen ( l ) ,however, recently reviewed the status quo in the field of FIA and concluded that "the theory of FLA, unfortunately, is still at a rather lamentable level compared with the sophisticated level of many of the practical developments. The concept of controlled dispersion, based on the dispersion coefficient, is a useful tool for the rational design of a flow system and for comparison and scaling of channels in FIA, yet it does not describe the response in a comprehensive fashion." There have been numerous attempts to derive a general expression to describe concentration as a function of time in a flow injection system. Historically, Taylor (2, 3) was the first to quantitatively treat the concept of axial dispersion occurring in straight, open tubes. Taylor's solutions of the diffusion-convection equation are most accurate for regions of flow where convection (high flow rates) or diffusion (low flow rates) is the dominant contribution to dispersion. Flow rates commolily employed in FIA result in conditions that are intermediate between these two limiting regions and are described by laminar flow. Dispersion in FIA is a consequence of both convective transport (axial direction) and diffusional transport (axial and radial directions) of sample molecules 0 1988 American Chemical Society
