BIOLOGICAL TRACE ELEMENT RESEARCH 4, 29-34 (1982)
Platelet Selenium in Children with Normal and Low Selenium Intake K.
KASPEREK, 1 I. LOMBECK, 2 J. KIEM,J, *
G. V.
IYENGAR, 1
Y . X . W A N G , 1 L . E . F E I N E N D E G E N , 1 AND H . J. BREMER 2
1Institute of Medicine, Nuclear Research Center, Box 1913, D-5170 Jiilich, Federal Republic of Germany and 2University Children's Hospital C, Moorenstr. 5, D-4000 Diisseldorf, Federal Republic of Germany Received August 11, 1981; Accepted August 31, 1981
The concentration of selenium was determined by instrumental neutron activation analysis in erythrocytes, platelets, and plasma of eight dietetically treated children with phenylketonuria (n = 6) or maple-syrup-urine disease (n = 2) with low selenium intake and for ten children with normal selenium intake. The normal selenium concentration in platelets was about 600 ng/g and about five times higher than in erythrocytes of the same children. A decreased selenium concentration in platelets was seen only when the corresponding concentrations in erythrocytes and plasma were very low. This suggests a special role of selenium in platelets. Index Entries: Selenium, in platelets; platelet selenium; erythrocytes, selenium in; plasma, selenium in; selenium deficiency, normal versus low selenium intake; children, Se dietotherapy of; phenylketonuria, Se concentration and; maple-syrupurine disease, Se concentration and.
Introduction In an earlier communication (1) we reported that the selenium concentration in platelets exceeds that of any hitherto examined body tissue. With the present investigation we wanted to compare the selenium concentration of platelets with those of erythrocytes and plasma, as recently reported (2, 3).
9 1982 by The Humana Press Inc. All fights of any nature whatsoever reserved. O1634984/82/0300-002952
.0 0
29
30
KASPEREK ET AL.
Materials and Methods From ten children aged 1.5 to 9.5 years (mean = 4.7) about 10 mL of citrated venous blood were taken between 8 and 9 AMbefore breakfast, together with aliquots for routine analysis before minor operations, e.g., uncomplicated inguinal hernia, or after recovery from slight infections. From eight patients aged 2.5 to 7.8 years (mean = 4.3) who were treated by approved dietary methods because of phenylketonuria (PKU) or maple-syrup-urine disease (MSUD), similar blood samples were taken during routine control of their amino acid metabolism. Their protein intake consisted mainly of protein hydrolyzates or amino acid mixtures. These eight patients were supplemented by trace elements, but usually selenium was not added (4, 5). Therefore, their daily selenium intake was low. Each of these 18 blood samples permitted preparation of two samples of erythrocytes and two of plasma, and also allowed preparation of an unwashed platelet pellet. The erythrocyte samples were not washed and therefore included 0.23 +- 0.10 g/g of plasma, as estimated by their iron concentrations. The plasma samples contained about 0.20 g/g of citrate anticoagulant. Both erythrocyte and plasma samples were not corrected for enclosed plasma or anticoagulant. The concentrations of selenium and other trace elements, e.g., iron, were determined in these 72 samples as described elsewhere (6). Only the data for selenium are reported here. In order to obtain platelet samples of sufficient size, the technique adopted in earlier experiments (1, 7, 8) had to be modified. Therefore, the platelet pellets were prepared and defined in a manner similar to that described elsewhere (9), with the following two modifications: 1. Samples containing 3.5 mL of platelet rich plasma (PRP) were obtained instead of 2 mL, thereby introducing considerable contamination from erythrocytes and leukocytes. The estimated mass fractions were 0.06 g erythrocytes and 0.02 g leukocytes per 1 g platelets (mean, n = 18). 2. 13q-Human serum albumin (13q-HSA, 0.03 mCi/mg HSA, AmershamBuchler, 3300 Braunschweig, F.R.G.) was used to correct for trapped plasma. The so-called "wall effect" (9) was determined for each of the 18 samples using platelet poor plasma (PPP). Since it was found to be 1.2 --- 0.4 mg (mean --- SD, n = 18) the average value of 1.2 mg was used to calculate the results of all the 18 platelet samples. The 18 dried plasma-containing platelet pellets were used for instrumental neutron activation analysis as previously described (1, 6) with the following modifications: 1. They were irradiated for 10 days. 2. After cooling with liquid nitrogen, the sealed quartz tubes were cut into two approximately equal pieces. The quartz piece containing the platelet pellet was filled with 0.5 mL of 70% nitric acid and incubated for 12 h at 50~ After carefully pipetting the dissolved platelet pellet into a glass tube, the quartz piece was refilled for 12 more hours with 1 mL of 70% nitric acid. This 1 mL was then also
31
PLATELET SELENIUM
transferred to the corresponding glass tube using the same pipet as for the corresponding 0.5 mL. 3. The results were calculated according to the formula: A = (B-
CD)/E
where A = concentration of selenium (ng/g) in plasma-free platelets on a wet weight basis (without correction for contaminating erythrocytes and leukocytes); B = amount of selenium (ng) in the platelet pellet; C = fraction of plasma (g) in the quartz tube containing the platelet pellet minus 0.002 g since we found in an additional experiment that, on average, about 2 mg of plasma sitting along the wall of the quartz tube was not washed out by the nitric acid procedure; D = concentration of selenium (ng/g) in plasma on a wet weight basis; E = fraction of platelets (g) in the platelet pellet.
Results Specification of the platelet pellets (mean --- SD, n = 18): platelet number per IxL of PRP by microscopy = 390,000 --- 158,000; platelet number per wL of PRP by the Coulter Counter = 381,000 -+ 140,000, total platelet number (• 109) in the platelet pellet = 0.995 - 0.320, mean platelet volume by the Coulter Counter = 6.6 -+ 0.6 fL, wet weight of the platelet fraction in the platelet pellet = 8.4 --- 2.5 mg, wet weight of the plasma fraction in the platelet pellet = 11.7 -+ 2.5 mg, mean platelet wet weight = 8.6 - 1.2 pg. Figure 1 shows the concentrations of selenium on a wet weight basis for erythrocytes, platelets, and plasma from 18 corresponding subjects arranged in a descending sequence of selenium concentration in erythrocytes. The platelets have about five times higher concentrations of selenium than erythrocytes for the subjects represented on the left side of Fig. 1. Although the concentrations of selenium in erythrocytes and plasma show a parallel continuously decreasing tendency, those in platelets decrease significantly only for the last seven subjects represented on the right side of Fig. 1. It is also interesting that the ratio between the lowest and normal selenium concentration is about 1/4 for platelets, whereas it is only about 1/7 for erythrocytes and 1/15 for plasma. Figure 2 shows that the concentrations of selenium were significantly lower [P(t) > 0.999] in platelets from patients under dietotherapy (right side) than from the control children (left side).
Discussion In order to base the results on the wet weight of unwashed plasma-free platelets (1, 8, 9), the weight of the platelet fraction and that of the plasma fraction in the platelet pellet have to be determined. Since this is difficult for small samples, we obtained additional data as control parameters (see Results). They are in good agree-
32
KASPEREK ET AL.
Concentrotion of selenium on wet weight basis (ng/g)
100 ~
erythrocytes
I iiiiiiii!!!!ii i iiii!"i''i
m n n n ~ ~ i ii i ii
=
O
......
-'Ill -=II I
=, __= - , , = , 1 1 I=,=,
500 l 0 a-
18 corresponding subjects arranged in a descending sequence of selenium concentration in erythrocytes Fig. 1. Concentrations of selenium on a wet weight basis for erythrocytes, platelets, and plasma from 18 corresponding subjects. ment with corresponding data recently obtained for larger platelet samples (9) if the methodological differences are taken into account. This proves that the 18 platelet samples were well controlled. The selenium concentrations in platelets obtained for children without dietotherapy (Fig. 2), which were of the order of 600 ng/g, cannot directly be compared with those obtained previously for adults (1) since platelet collection and sample preparation were different. The results shown in Fig. 1 prove that the concentration of selenium is lowered in platelets when the concentration of selenium is also lowered considerably in erythrocytes and plasma. This can be the case in dietetically treated patients with
33
PLATELET SELENIUM
i
v
600 fl;
| !
0 13. r~
controls ! r-
.~ 200 !
dietotherapy
r CP 0
_
(..)
Fig. 2. Platelet selenium concentrations of 8 patients under dietotherapy (right side) and 10 control subjects (left side). PKU or MSUD (Fig. 2). But it seems that the platelets tend to retain their selenium very firmly even in cases where the selenium concentration is already lowered in erythrocytes and plasma (Fig. 1). This signals further the importance of selenium to platelets.
Acknowledgment This study was supported by the "Deutsche Forschungsgemeinschaft." Dr. Wang is supported by the "A.v. Humboldt-Stiftung".
References 1. K. Kasperek, G. V. Iyengar, J. Kiem, H. Borberg, and L. E. Feinendegen, Clin, Chem. 25, 711 (1979).
34
K A S P E R E K ET A L .
2. J. Kiem, G. V. Iyengar, K. Kasperek, I. Lombeck, Y. X. Wang, and L. E. Feinendegen, In Proceedings of the Nordic Symposium on Mineral Elements '80, Espoo, Finland, 1980, in press. 3. I. Lombeck, K. Kasperek, G. V. Iyengar, J. Kiem, L. E. Feinendegen, and H. J. Bremer, In Proceedings of the Fourth International Symposium on Trace Element Metabolism in Man and Animals, Perth (Australia), 1981, in press. 4. I. Lombeck, K. Kasperek, H. D. Harbisch, L. E. Feinendegen, and H. J. Bremer, Eur. J. Pediatr. 125~ 81 (1977). 5. I. Lombeck, K. Kasperek, H. D. Harbisch, K. Becker, E. Schumann, W. Schr6ter, L. E. Feinendegen, and H. D. Bremer, Eur. J. Pediatr. 128, 213 (1978). 6. K. Kasperek, In Proceedings of the Second lnternational Conference on Nuclear Methods in Environmental Research, U.S. Energy Research and Development Administration, Conf. 740701, Univ. of Missouri, Columbia, MO, 1974, p. 140. 7. G. V. Iyengar, H. Borberg, K. Kasperek, J. Kiem, M. Siegers, L. E. Feinendegen, and R. Gross, Clin. Chem. 25, 699 (1979). 8. J. Kiem, H. Borberg, G. V. Iyengar, K. Kasperek, M. Siegers, L. E., Feinendegen, and R. Gross, Clin. Chem. 25, 705 (1979). 9. J. Kiem, H. Weese, Y. X. Wang, and L. E. Feinendegen, Clin. Chem., submitted.
Platelet Selenium in Children with Normal and Low Selenium Intake K.
KASPEREK, 1 I. LOMBECK, 2 J. KIEM,J, *
G. V.
IYENGAR, 1
Y . X . W A N G , 1 L . E . F E I N E N D E G E N , 1 AND H . J. BREMER 2
1Institute of Medicine, Nuclear Research Center, Box 1913, D-5170 Jiilich, Federal Republic of Germany and 2University Children's Hospital C, Moorenstr. 5, D-4000 Diisseldorf, Federal Republic of Germany Received August 11, 1981; Accepted August 31, 1981
The concentration of selenium was determined by instrumental neutron activation analysis in erythrocytes, platelets, and plasma of eight dietetically treated children with phenylketonuria (n = 6) or maple-syrup-urine disease (n = 2) with low selenium intake and for ten children with normal selenium intake. The normal selenium concentration in platelets was about 600 ng/g and about five times higher than in erythrocytes of the same children. A decreased selenium concentration in platelets was seen only when the corresponding concentrations in erythrocytes and plasma were very low. This suggests a special role of selenium in platelets. Index Entries: Selenium, in platelets; platelet selenium; erythrocytes, selenium in; plasma, selenium in; selenium deficiency, normal versus low selenium intake; children, Se dietotherapy of; phenylketonuria, Se concentration and; maple-syrupurine disease, Se concentration and.
Introduction In an earlier communication (1) we reported that the selenium concentration in platelets exceeds that of any hitherto examined body tissue. With the present investigation we wanted to compare the selenium concentration of platelets with those of erythrocytes and plasma, as recently reported (2, 3).
9 1982 by The Humana Press Inc. All fights of any nature whatsoever reserved. O1634984/82/0300-002952
.0 0
29
30
KASPEREK ET AL.
Materials and Methods From ten children aged 1.5 to 9.5 years (mean = 4.7) about 10 mL of citrated venous blood were taken between 8 and 9 AMbefore breakfast, together with aliquots for routine analysis before minor operations, e.g., uncomplicated inguinal hernia, or after recovery from slight infections. From eight patients aged 2.5 to 7.8 years (mean = 4.3) who were treated by approved dietary methods because of phenylketonuria (PKU) or maple-syrup-urine disease (MSUD), similar blood samples were taken during routine control of their amino acid metabolism. Their protein intake consisted mainly of protein hydrolyzates or amino acid mixtures. These eight patients were supplemented by trace elements, but usually selenium was not added (4, 5). Therefore, their daily selenium intake was low. Each of these 18 blood samples permitted preparation of two samples of erythrocytes and two of plasma, and also allowed preparation of an unwashed platelet pellet. The erythrocyte samples were not washed and therefore included 0.23 +- 0.10 g/g of plasma, as estimated by their iron concentrations. The plasma samples contained about 0.20 g/g of citrate anticoagulant. Both erythrocyte and plasma samples were not corrected for enclosed plasma or anticoagulant. The concentrations of selenium and other trace elements, e.g., iron, were determined in these 72 samples as described elsewhere (6). Only the data for selenium are reported here. In order to obtain platelet samples of sufficient size, the technique adopted in earlier experiments (1, 7, 8) had to be modified. Therefore, the platelet pellets were prepared and defined in a manner similar to that described elsewhere (9), with the following two modifications: 1. Samples containing 3.5 mL of platelet rich plasma (PRP) were obtained instead of 2 mL, thereby introducing considerable contamination from erythrocytes and leukocytes. The estimated mass fractions were 0.06 g erythrocytes and 0.02 g leukocytes per 1 g platelets (mean, n = 18). 2. 13q-Human serum albumin (13q-HSA, 0.03 mCi/mg HSA, AmershamBuchler, 3300 Braunschweig, F.R.G.) was used to correct for trapped plasma. The so-called "wall effect" (9) was determined for each of the 18 samples using platelet poor plasma (PPP). Since it was found to be 1.2 --- 0.4 mg (mean --- SD, n = 18) the average value of 1.2 mg was used to calculate the results of all the 18 platelet samples. The 18 dried plasma-containing platelet pellets were used for instrumental neutron activation analysis as previously described (1, 6) with the following modifications: 1. They were irradiated for 10 days. 2. After cooling with liquid nitrogen, the sealed quartz tubes were cut into two approximately equal pieces. The quartz piece containing the platelet pellet was filled with 0.5 mL of 70% nitric acid and incubated for 12 h at 50~ After carefully pipetting the dissolved platelet pellet into a glass tube, the quartz piece was refilled for 12 more hours with 1 mL of 70% nitric acid. This 1 mL was then also
31
PLATELET SELENIUM
transferred to the corresponding glass tube using the same pipet as for the corresponding 0.5 mL. 3. The results were calculated according to the formula: A = (B-
CD)/E
where A = concentration of selenium (ng/g) in plasma-free platelets on a wet weight basis (without correction for contaminating erythrocytes and leukocytes); B = amount of selenium (ng) in the platelet pellet; C = fraction of plasma (g) in the quartz tube containing the platelet pellet minus 0.002 g since we found in an additional experiment that, on average, about 2 mg of plasma sitting along the wall of the quartz tube was not washed out by the nitric acid procedure; D = concentration of selenium (ng/g) in plasma on a wet weight basis; E = fraction of platelets (g) in the platelet pellet.
Results Specification of the platelet pellets (mean --- SD, n = 18): platelet number per IxL of PRP by microscopy = 390,000 --- 158,000; platelet number per wL of PRP by the Coulter Counter = 381,000 -+ 140,000, total platelet number (• 109) in the platelet pellet = 0.995 - 0.320, mean platelet volume by the Coulter Counter = 6.6 -+ 0.6 fL, wet weight of the platelet fraction in the platelet pellet = 8.4 --- 2.5 mg, wet weight of the plasma fraction in the platelet pellet = 11.7 -+ 2.5 mg, mean platelet wet weight = 8.6 - 1.2 pg. Figure 1 shows the concentrations of selenium on a wet weight basis for erythrocytes, platelets, and plasma from 18 corresponding subjects arranged in a descending sequence of selenium concentration in erythrocytes. The platelets have about five times higher concentrations of selenium than erythrocytes for the subjects represented on the left side of Fig. 1. Although the concentrations of selenium in erythrocytes and plasma show a parallel continuously decreasing tendency, those in platelets decrease significantly only for the last seven subjects represented on the right side of Fig. 1. It is also interesting that the ratio between the lowest and normal selenium concentration is about 1/4 for platelets, whereas it is only about 1/7 for erythrocytes and 1/15 for plasma. Figure 2 shows that the concentrations of selenium were significantly lower [P(t) > 0.999] in platelets from patients under dietotherapy (right side) than from the control children (left side).
Discussion In order to base the results on the wet weight of unwashed plasma-free platelets (1, 8, 9), the weight of the platelet fraction and that of the plasma fraction in the platelet pellet have to be determined. Since this is difficult for small samples, we obtained additional data as control parameters (see Results). They are in good agree-
32
KASPEREK ET AL.
Concentrotion of selenium on wet weight basis (ng/g)
100 ~
erythrocytes
I iiiiiiii!!!!ii i iiii!"i''i
m n n n ~ ~ i ii i ii
=
O
......
-'Ill -=II I
=, __= - , , = , 1 1 I=,=,
500 l 0 a-
18 corresponding subjects arranged in a descending sequence of selenium concentration in erythrocytes Fig. 1. Concentrations of selenium on a wet weight basis for erythrocytes, platelets, and plasma from 18 corresponding subjects. ment with corresponding data recently obtained for larger platelet samples (9) if the methodological differences are taken into account. This proves that the 18 platelet samples were well controlled. The selenium concentrations in platelets obtained for children without dietotherapy (Fig. 2), which were of the order of 600 ng/g, cannot directly be compared with those obtained previously for adults (1) since platelet collection and sample preparation were different. The results shown in Fig. 1 prove that the concentration of selenium is lowered in platelets when the concentration of selenium is also lowered considerably in erythrocytes and plasma. This can be the case in dietetically treated patients with
33
PLATELET SELENIUM
i
v
600 fl;
| !
0 13. r~
controls ! r-
.~ 200 !
dietotherapy
r CP 0
_
(..)
Fig. 2. Platelet selenium concentrations of 8 patients under dietotherapy (right side) and 10 control subjects (left side). PKU or MSUD (Fig. 2). But it seems that the platelets tend to retain their selenium very firmly even in cases where the selenium concentration is already lowered in erythrocytes and plasma (Fig. 1). This signals further the importance of selenium to platelets.
Acknowledgment This study was supported by the "Deutsche Forschungsgemeinschaft." Dr. Wang is supported by the "A.v. Humboldt-Stiftung".
References 1. K. Kasperek, G. V. Iyengar, J. Kiem, H. Borberg, and L. E. Feinendegen, Clin, Chem. 25, 711 (1979).
34
K A S P E R E K ET A L .
2. J. Kiem, G. V. Iyengar, K. Kasperek, I. Lombeck, Y. X. Wang, and L. E. Feinendegen, In Proceedings of the Nordic Symposium on Mineral Elements '80, Espoo, Finland, 1980, in press. 3. I. Lombeck, K. Kasperek, G. V. Iyengar, J. Kiem, L. E. Feinendegen, and H. J. Bremer, In Proceedings of the Fourth International Symposium on Trace Element Metabolism in Man and Animals, Perth (Australia), 1981, in press. 4. I. Lombeck, K. Kasperek, H. D. Harbisch, L. E. Feinendegen, and H. J. Bremer, Eur. J. Pediatr. 125~ 81 (1977). 5. I. Lombeck, K. Kasperek, H. D. Harbisch, K. Becker, E. Schumann, W. Schr6ter, L. E. Feinendegen, and H. D. Bremer, Eur. J. Pediatr. 128, 213 (1978). 6. K. Kasperek, In Proceedings of the Second lnternational Conference on Nuclear Methods in Environmental Research, U.S. Energy Research and Development Administration, Conf. 740701, Univ. of Missouri, Columbia, MO, 1974, p. 140. 7. G. V. Iyengar, H. Borberg, K. Kasperek, J. Kiem, M. Siegers, L. E. Feinendegen, and R. Gross, Clin. Chem. 25, 699 (1979). 8. J. Kiem, H. Borberg, G. V. Iyengar, K. Kasperek, M. Siegers, L. E., Feinendegen, and R. Gross, Clin. Chem. 25, 705 (1979). 9. J. Kiem, H. Weese, Y. X. Wang, and L. E. Feinendegen, Clin. Chem., submitted.
