Jan 18, 1989 - green method (11) in a C-300 system (Greiner AG, Langen- thai, Switzerland). ... was pressed to one of the empty bags in the blood- collecting ...
CLIN. CHEM. 35/4, 537-540 (1989)
AtomicAbsorptionSpectrometricDeterminationof Selenium in Human Blood Components Lena Hansson,’ Jean Pettersson,1
Lars Erlksson,2
and Ake Olin1
We separated blood from five healthy blood donors into
Reagents
plasma, erythrocytes, platelets, and leukocytes; counted the number of cells in each fraction; and determined the selenium content of each component by hydride generation atomic absorption spectrometry. The mean (± SD) selenium concentrations and amounts measured were as follows: whole blood 102.3 ± 16.1 ig/L, plasma 76.9 ± 10.6 ug/L, erythrocytes 13.7 ± 2.8 ag per cell, platelets 4.8 ± 1.1 ag per cell, and leukocytes 99 ± 26 ag per cell.
All reagents and chemicals were of “pro analysi” quality, and the water used was de-ionized, distilled, and filtered through a Milli-Q-system (Millipore, Bedford, MA).
The role of selenium as an essential element in humans has been discussed by several workers, (e.g., 1-3), and its deficiency is the probable cause of several diseases, Keshan disease (4) perhaps being the most striking example. Several reports provide information about the selenium concentrations in serum, plasma, or whole blood (e.g., 5-7) and in erythrocytes (e.g., 8). However, few have dealt with the concentrations of selenium in human platelets or leukocytes
prepared only washed and unwashed platelet concentrates. These and their corresponding acidified supernatant plasmas (ASP) were analyzed for selenium and albumin. Albumin was also determined in the platelet concentrates and ASP from bloods 1-5. The separations were carried out as follows. We mixed 450 mL of blood with 63 mL of citrate-phosphate--dextrose anticoagulant (CPD) in a quadruple blood-bag system (Terumo Corp., Tokyo, Japan) (13) containing CPD and salineadenine-glucose-mannitol (SAGM). The composition of CPD is trisodium citrate dihydrate, 26.30 g/L; citric acid monohydrate, 3.27 g/L; sodium dihydrogen phosphate monohydrate, 2.22 g/L; and dextrose, 25.50 g/L. That of the SAGM solution is sodium chloride, 8.77 g/L; adenine, 0.169 g/L; glucose, 8.18 g/L; and mannitol, 5.25 g/L. For the analysis of whole blood, 10 mL of this was withdrawn into a test tube. The remaining sample in the blood bag was centrifuged for 1.5 miii at 2240 x g (14). The platelet-rich plasma was pressed to one of the empty bags in the bloodcollecting system and that bag was sealed off. The plateletrich plasma was acidified with acid-citrate-dextrose anticoagulant (ACD; Travenol Laboratories, Norfolk, U.K.), 10 mL per 100 g of platelet-rich plasma (15). The composition of ACD is trisodium citrate dihydrate, 22 g/L; citric acid monohydrate, 8 g/L; and dextrose, 22.4 g/L. The acidified platelet-rich plasma was centrifuged for 10 mm at 3300 X g and all plasma was pressed from the bag. This plasma was analyzed in the “trapped plasma” study and is referred to as ASP. The platelet pellet was resuspended in a platelet storage medium composed of potassium chloride, 0.75 g/L; disodium hydrogen phosphate dihydrate, 0.89 gfL; trisodium citrate dihydrate, 8.82 gIL; sodium chloride, 4.09 g/L; r-mannitol, 5.47 g/L; and glucose, 0.9 g/L (L. Briksson, in preparation) for specimens 1-5. We suspended specimens A-D in the same medium, but added Na2EDTA, 1.67 g/L; theophylline, 0.18 g/L; and prostaglandin E1, 33 g/L to prevent leakage from the platelets in the subsequent washings (16). We attached an empty 150-mL transfer bag (Transfer Pack 4R2001; Fenwal Laboratories, Deerfield, IL) to the blood-bag system, using a sterile connecting device (SCD 312; Haemonetic Corp., Braintree, MA), then centrifuged the blood-bag system for 10 miii at 3300 x g. The plateletpoor plasma was pressed to the empty 300-mL transfer bag and the buffy coat to the 150-mL transfer bag. We added SAGM solution to the erythrocyte fraction. Purification of the blood components. The platelet-poor plasma was recentrifuged for 10 mm at 3300 x g to obtain the plasma sample.
(9). Here we have determined selenium in whole blood, plasma, erythrocytes, platelets, and leukocytes from healthy Swedish blood donors. The blood cells were separated from whole blood by centrifugation and the number of each cell type was counted in all fractions, including the original specimen. Total selenium was determined in each fraction by hydride generation atomic absorption spectrometry (HGAAS) after destruction of the organic matter.3
Materials and Methods Apparatus The blood components were separated with an IEC Damon DPR-6000 centrifuge (International Bquipment Co., Needham Heights, MA). The concentrations of cells in the various fractions were counted in a Technicon H-i system (Technicon Instruments Corp., Tarrytown, NY). The apparatus for the digestion of samples and the HG-AAS instrumentation have been described elsewhere (10). Albumin in plasma was determined with a bromcresol green method (11) in a C-300 system (Greiner AG, Langenthai, Switzerland). In the platelet fractions, albumin was quantified with an immunological rabbit anti-albumin method (12) modified to a turbidinietric method in a Multistat ifi Plus centrifugal analyzer system (Instrumentation Laboratory,
Lexington,
MA).
‘Department of Analytical Chemistry, University of Uppsala, P.O. Box 531, S-751 21 Uppsala, Sweden. 2Department of Clinical Immunology and Transfusion Medicine, University Hospital, S-751 85 Uppsala, Sweden. 3Nonstandard abbreviations: HG-AAS, hydride generation atomic absorption spectrometry; CPD, citrate-phosphate-dextrose anticoagulant; SAGM, saline-adenine-glucose-mannitol; ASP, acidified supernatant plasma; and ACD, acid-citrate-dextrose anticoagulant. Received November 16, 1988; accepted January
18, 1989.
Procedures Collection and separation of blood. Blood specimens were taken from nine donors. Five of the specimens, 1-5, were fractionated as described below, and all fractions were analyzed for selenium. From the other four, A-D, we
CLINICALCHEMISTRY, Vol. 35, No.4, 1989 537
The resuspended platelet concentrate was transferred to polyvinyl chloride test tubes and centrifuged for 2.5 mm at 295 x g. We recentrifuged the supernate for 5 mm at 165 x g and used this as the platelet fraction. For samples A-D, we split this fraction into two halves, analyzing one without further purification, and washing the other twice before resuspending it in the platelet medium described above, then analyzing. We recentrifuged the erythrocyte suspension for 10 mm at 3300 x g, then pressed the supernate and the “new” buffy coat into an empty bag and discarded them. Then we washed the erythrocytes twice with 300-mL portions of isotonic saline (NaC1 9 g/L) solution containing 5 mmol of glucose per liter and proceeded with analysis. We purified the leukocyte fraction by the method of H#{226}kansson and Venge (17), but used the saline-glucose solution instead of Gey’s buffer. Selenium determination. On the same day as the separation procedures, we weighed appropriate amounts of sample into 50-mL glass tubes, digested these by the “magnesium nitrate method,” and determined the selenium content by HG-AAS (18). All samples were analyzed in duplicate. The sample sizes were as follows: whole blood or erythrocyte fraction, 0.5 g; plasma, 1 g; platelet fraction, >5 x iO cells; leukocyte fraction, >2.5 x 10 cells. Because we used a weighed sample for the selenium determination and recorded the number of cells per volume with the cell counter, we could convert the sample mass to the corresponding volume by dividing by the density, which was determined by
be altered. In addition, the risk for platelet agglomeration increases, which may lead to an erroneous count of the number of cells in the fractions. On the other hand, if not washed, plasma will be retained on and between the cells. This will contribute to the measured trace-element content, and has to be corrected for. We estimated the amount of trapped plasma in the platelet fractions by determining albumin in the platelet concentrates and in their ASP. Then a correction for trapped plasma selenium could be made from a knowledge of the selenium concentration in the ASP, and the assumption that all albumin derives from the ASP. We found that about 45% of the selenium content of the unwashed fractions was present in trapped plasma. To check the correction based on the albumin determinations, we split the platelet fractions from specimens A-D into two parts. One part was washed; the other was not. Selenium and albumin were determined in washed and unwashed platelet concentrates, and in their respective ASP. Table 1 shows the analytical data obtained. These data were used to calculate the amounts of selenium in the platelets as presented in Table 2. The results for the washed platelets and the unwashed platelets corrected for plasma selenium are consistent, which demonstrates the validity of this correction method. It can also be concluded that the washing of the platelets did not cause any detectable leakage of
weighing
Whole blood and plasma. We found the selenium concentration in whole blood from donors 1-5 to be 102.3 ± 16.1 pg/L. The corresponding value for plasma was 76.9 ± 10.6 zg/L. These values accord with other reports (6-8), but it must be borne in mind that local variations may be considerable, owing to differences in abundance of the element between different geographical areas of the world. The linear correlation coefficient between the selenium concentrations in plasma and whole blood was 0.98. Blood cells. For each separated specimen (1-5) of whole blood we calculated the selenium contents of the three cell types from mass balance equations based on the analytical results for the erythrocyte, platelet, and leukocyte fractions, viz.:
aliquots
in 2- to l0-mL
measuring
flasks.
selenium.
Analytical Results
The
plasma and whole-blood samples had densities of 1.023 g/ mL and 1.047 g/mL, respectively. The density of the erythrocyte fraction was 1.08 g/rnL, and that of the leukocyte and the platelet fractions was near 1.00.
Results and Discussion Sample size. The sample size required for selenium determination in whole blood, plasma, or packed erythrocytes is quite moderate. A sample weighing 0.5 g suffices to generate an atomic absorption signal large enough to yield a CV of about 2% in the analytical result (18). The number of platelets and leukocytes in blood is small, requiring their collection from a relatively large volume of blood. If a CV of 5% or less is desired for the determination, one can calculate from the amount of selenium in leukocytes that about 5’ 108 cells are required for duplicate measurement of samples diluted to 25 mL after the digestion step. This number of cells is present in about 100 mL of whole blood. For platelets the corresponding figures are 1 1010 cells and 50 mL of blood. The actual volume required will depend on the effectiveness of the separation scheme. For this work we used 450 mL of blood, so as to obtain enough leukocytes. Trapped plasma. Platelets can easily be damaged, even by washing with isotonic solutions, unless special precautions are taken (19). Their content of trace elements may thereby
C = nx C is a column concentrations
vector containing the measured selenium (tg/L) in the three fractions, ii is a 3’3 matrix containing the corresponding number of cells of each type per liter, and x is a column vector of the unknown selenium content in each kind of cell (tg). The selenium concentration in the platelet fractions was corrected for trapped plasma as estimated from the albumin determinations. See Table 3 for an example of data used in these calculations and note that the purity of the fractions with respect to cell type leads to a well-conditioned set of equa-
Table 1. Plasma Selenium Trapped In Platelet Co ncentrates Supernatant plasma
Wash ed platelet concn
Unwashed platelet concn
Donor
Albumin, g/L
Se, ig/L
Albumin, g/L
Se, cg/L
Platelets’
Albumin, g/L
Se, g/L
Platelets’
A B C D
36.2 36.3 37.6 37.7
50.3 69.6 56.1 62.0
3.8 4.9 3.8 4.1
13.0 18.5 13.6 17.4
1764 1485 1788
0.010 0.003 0.003
1434
0.002
6.75 6.04 5.20 6.96
1749 957 1209 1113
‘Numbe r divided by iO
538
CLINICAL CHEMISTRY, Vol. 35, No. 4, 1989
Table 2. Selenium Content of Platelets Calculated from the Data in Table 1 Selenium, ag (10_la g) per platelet
Unwashed Donor
platelets
corrected for plasma Se
Unwashedplatelets Donor
3.9
4.4
1
7.4
B
12.5
6.3
6.1
C D
7.6 12.1
4.3 6.3
4.4 7.4
.
Table 3. Number of Cells per Liter, and Selenium Concentration (p.gIL) in Whole Blood and Its Fractions from One Donor .1Q_12
blood Plasma Erythrocyte
3.75
Platelets,
Leukocytes,
10’#{176}
.
.
22.2 1.00
0.00
Se concn,
10
6.17 0.01 9.70 0.20 0.06 Platelet 0.00 284 0.10 Leukocyte 0.07 1.30 98.4 ‘14.1 g/L after correction for trappedplasmaselenium.
Whole
Calculated
92.7 72.0 115.1 24.18
9.1
Difference -1.1
Measured
91.6
92.7
126.2 85.1 107.8 95.9
127.1 85.4 108.0 98.2
#{149}
2 3 4 5
tions. The results, in attograms of selenium per cell, are presented in Table 4. When the erythrocyte mean value, 13.7 ag per cell, is converted to a microgram per liter packed-cell value by multiplication by the mean number of erythrocytes per liter (3.61 10) and division by the mean hematocrit (0.34) the result is 145.5 tg/L. This agrees with the results of others (8). The mean selenium content of the platelets is 4.8 ag per cell. Including the platelet results from samples A-D, we obtain a mean of 5.2 ± 1.1 ag per cell. If we assume a mean platelet weight of 10 pg (19), we arrive at 520 ng/g, which can be compared with the 782 ng/g obtained by Kasperek et al. (9). We have found very little information in the literature about the selenium concentration in leukocytes. Determination of selenium in individual leukocytes has been performed (20), but these measurements were mainly made on individuals supplemented with selenium. In addition, the results were given in micrograms per gram dry substance and thus they are difficult to compare with our mean value of 99 ag per cell. However, the result of Johansson et al. (20) for leukocytes has been recalculated to obtain a value expressed per cell (U. Lindh, personal comm.). A range of about 200-700 ag per cell was obtained for neutrophil granulocytes from an individual supplemented with 200 p.g of sodium selenite per day for two months. The normally-
Erythrocytes,
Selenium concn, zg/L
Washed platelets
A
FractIon
Table 5. Measured Concentration of Selenium In Whole Blood and the Calculated Sum of the Different Components
-0.9 -0.3 -0.2 -2.3
present concentrations of selenium could ordinarily not be measured by the method used by Johansson et al. (20). Our result refers to an average value for a mixture of a large number of all types of leukocytes. In the leukocyte fractions, about 70% of the cells were neutrophil granulocytes. Method control. For method control, we summed the contributions
to the
whole-blood
selenium
concentration
from the different fractions, and compared the sum with the measured concentration. The calculated concentration was obtained from [Selcaic
= [Seli88
1T%pla
+ Xe
11e +
Xplat
nplat
+ x1 ‘n1
v%1 is the volume fraction of plasma in the whole blood calculated from the hematocrit value; [Se] represents selenium concentrations in micrograms per liter; x is the average selenium content of a single cell (pg); and n the number of such cells per liter in whole blood. The subscripts refer to plasma, erythrocytes, platelets, and leukocytes, respectively. If no systematic errors were present, the calculated and measured concentrations should be equal. Table 5 gives the differences between these values for blood specimens 1-5. The mean of the differences is -1.0 ± 1.0 gIL (95% confidence limits). This indicates the absence of substantial systematic errors in the sampling and separation procedures, the counting of the cells, the sample workup, and final determination of selenium as far as whole blood, plasma, and erythrocyte fractions are concerned. For the platelets and leukocytes it is impossible to discover even large systematic errors because of their small contributions to the total blood selenium concentration. Our results indicate that, on the average, less than 1% of the selenium in human whole blood originates from platelets and less than 0.5% from leukocytes. where
References 1. Shamberger RJ. Antioxidation and trace elements in cancer. Epidemiologic review. In: Bostr#{246}m H, L4jungstedt N, eds. Trace elements in health and disease. Stockholm: Almqvist & Wiksell, 1985:155-71.
Table 4. Selenium Content of Erythrocytes, Platelets, and Leukocytes, and Concentration in Plasma Erythrocytes
Donor
Platelets
Leukocytes
Selenium, ag per cell
Plasma Se, tg/L
1 2 3 4 5
11.9 17.7 12.6 15.4 11.0
5.0
83
6.2 4.4
94 142
Mean
13.7
SD
2.7
72.0 92.1 63.4
4.5
74
79.9
4.0
100
77.1
4.8 0.9
99 26
76.9 10.6
2. Salonen JT. Selenium
in cardiovascular diseases and cancer. Epidemiologic findings from Finland. Ibid, 172-86. 3. Merts W. Newer trace elements in human and animal nutrition and health. In: Mineral elements ‘80, Proceedings, Part II. Helsinki: Hanasaari Culture Center, 1981:381-98. 4. Keshan Disease Research Group. Observations on effect of sodium selenite in prevention of Keshan disease. Chin Med J 1979;92:471-6. 5. Welz B, Melcher M, Schlemmer G. Determination of selenium in human blood serum. Comparison of two atomic-absorption spectrometric procedures. Z Anal Chem 1983;316:271-6. 6. Macpherson KM, Sampson B, Diplock AT. Comparison of methods for the determination of selenium in biological fluids. Analyst 1988;113:281-3. CLINICAL CHEMISTRY, Vol. 35, No. 4, 1989 539
7. Verlinden M. On the acid decomposition of human blood and plasma for the determination of selenium. Talanta 1982;29:875-82. 8. Wasowicz W, Zachara BA. Selenium concentrations in the blood
Vox Sang 1981;40:65-70. 15. Mourad N. A simple method for obtaining platelet concentrates free of aggregates. Transfusion 1968;8:48. 16. Sandberg H, Andersson L-O. A highly sensitive assay of platelet factor 3 using a chromogenic substrate. Thromb Rea
and urine of a healthy Polish sub-population. J Clin Chem Clin Biochem 1987;25:409-12. 9. Kasperek K, Iyengar GV, Kiem J, Borberg H, Feinendegen LE. 1979;14:113-24. Elemental composition of platelets. Part ifi. Determination of Ag, 17. Hbkansson L, Venge P. The influence of serum on random Au, Cd, Co, Cr, Cs, Mo, Rb, Sb, and Se in normal human platelets by migration and chemotaxis of polymorphonuclear leucocytes: methneutron activation analysis. Clin Chem 1979;25:711-5. odological evaluation using sera from infection-prone patients and 10. Pettersson J, Hansson L, Olin A. Comparison of four digestion normals. Scand J Immunol 1980;11:271-82. methods for the determination of selenium in bovine liver by 18. Hansson L, Pettersson J, Olin A. A comparison of two digestion hydride generation and atomic-absorption spectrometry in a flow procedures for the determination of selenium in biological material. system. Talanta 1986;33:249-54. Talanta 1987;34:829-33. 11. Doumas BT, Watson WB, Briggs HG. Albumin standards and 19. Kiem J, Iyengar GV, Borberg H, et al. Sampling and sample measurements of serum albumin with bromcresol green. Clin Chim preparation of platelets for trace element analysis and determinaActs 1971;31:87-96. tion of certain selected bulk and trace elements in normal human 12. Lizana J, Helising K. Polymer enhancement of automated platelets by means of neutron activation analysis. Nucl Act Tech immunological nephelometric analysis, as illustrated by determinaLife Sci, Proc hit Symp. Vienna, Austria: hit. Atomic Energy tion of urinary albumin. Clin Chem 1974;20:415-20. Agency, 1979:143-64. 13. Hogman CF, Hedlund K. Storage of red cells in a CPD/SAGM 20. Johansson E, Lindh U, Landstr#{246}m E. The incorporation of system using Teruflex PVC. Vox Sang 1985;49:177-80. selenium and alterations of macro- and trace element levels in individual blood cells following supplementation with sodium sele14. Eriksson L, Hogman CF, Busch C. Lack of conformity in the behaviour of platelets during normal storage conditions at 22 #{176}C.nite and vitamin E. Biol Trace Elem Res 1983;5:433-47.
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