@Copyright 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/1200-0101502.00
Interactions of Selenium with Metal Ions at the Cellular Level ERLAND JOHANSSON* AND ULF LINDH
Department of Physical Biology, Gustaf Wemer Institute, Uppsala University, Box 531, S- 751 21 Uppsala, Sweden ABSTRACT In a supplementation study in which organic selenium as l-selenomethionine was administered in low doses during 1 yr, alterations in the concentrations of metal ions in the erythrocytes and the neutrophil granulocytes were observed. In the erythrocytes, altered concentrations of zinc were parallel with selenium. The concentrations of magnesium, calcium, manganese, copper, and sulfur were not significantly altered. However, altered concentrations of iron and zinc were observed in the neutrophils. The concentrations of magnesium, calcium, manganese, copper, and sulfur were not significantly altered. The accumulation of selenium in individual blood cells was different from that obtained with supplementation of inorganic selenium. When organic selenium was supplemented, the thrombocytes accumulated more selenium than the erythrocytes and the neutrophil granulocytes. The observations indicate that selenium interacts with metal ions at the cellular level when supplemented in low doses. The chemical form of selenium might be important in nutrition and therapy in view of the interaction and distribution pattern at the cellular level. Index Entries" l-fielenomethionine; selenium; iron; zinc, calcium; trace elements; erythrocytes; thrombocytes; neutrophil granulocytes; metal ion interactions; nuclear microprobe; micro-PIXE.
INTRODUCTION The bioavailability of an element is a complex function of several factors. Among those are the chemical form of the element, uptake and *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1 01
VoL 12, 1987
102
Johansson and Lindh
transport kinetics, metabolic transformations, and genetic influence. There is no satisfactory model for the availability of trace elements accounting for interactions with other elements. To completely understand the mechanisms of bioavailability and the importance of interactions with other elements, it is essential to know the chemical form of the nutrient. Lack of analytical methods to assess the redox state and to determine the low concentrations has probably hampered the inclusion of the chemical form in the recommendations of dietary intake (RDA) of macro- and trace elements. It is therefore desirable to design experiments using trace elements in known chemical forms to cast light upon the concept of bioavailability. Using known chemical forms of trace elements facilitates identifications of interactions at the cellular level. This is one of the prerequisites for more precise models to assess the bioavailability of trace elements in humans. Selenium attracted an increasing amount of interest after the discovery of Keshan Disease (1,2) and Kashin Beck Disease (3), which appear to be the first documented human diseases with low selenium status as one important risk factor. In these studies, plasma selenium, whole blood selenium, and hair selenium were used to assess the selenium status. Blood cells with different life spans, however, might be more useful as a model (4) of bioavailability because the uptake and distribution of trace elements as well as interactions with other elements can be monitored using the nuclear microprobe (5). In addition, it is necessary to investigate the correlation between cell functions and trace element concentrations. Studies of the interactions of selenium with other elements at the cellular level are scarce. Studies by Lindh and Johansson (5), Johansson et al. (6), and Johansson and Lindh (7) showed that when inorganic selenium was supplied for I yr, interactions with other elements occurred at the cellular level, as reflected in the elemental profile of individual blood cells. When the selenium concentration was altered, some metal ions were altered in a regular manner. The concentrations of iron, copper, manganese, and zinc were altered in the neutrophil granulocytes, and the thrombocytes displayed altered concentrations of iron and zinc. Since l-selenomethionine is one form of selenium in food, e.g., wheat, we have supplied 1-selenomethionine in low doses to mimic physiological conditions. Early, and also long-term, effects of the distribution of an essential element and the interaction with other elements might be important when plain food is fortified with such an element.
MATERIAL AND METHODS Organic selenium (50 p~g Se/d) as l-selenomethionine was supplied per os (orally) to one volunteer (52 yr, 74 kg), who was apparently healthy and not subjected to medication, with one exception. During 10 Biological Trace Element Research
VoL 12, 1987
Interactions of Selenium with Metal Ions
103
d, from d 100 to 110, penicillin [3 tablets Fenoxypen (666 rag) 3 times a day] was administered to cure an acute infection. The study was completed in 1 yr. The concentration of selenium in Swedish plain food is low; the daily intake is estimated as 30 ~g selenium/d. Fish and liver was restricted to once or less a week.
Blood Sampling and Cell Separation Five milliliters of venus blood (EDTA anticoagulant) were drawn at the same time of day, 11 AM. The different types of blood cellwhich rated immediately after sampling. The blood cells were fractionated by centrifugation at different speeds. The erythrocytes were isolated at 150g, the neutrophils at 400g, and the thrombocytes at 1000g. After the separation the pellets were s u s p e n d e d in 0.32M sucrose before disen, ing on backings of thin Formvar film. The cells were then quench frozled freeze-dried, and stored in a desiccator before analysis. A more detai KV, 15~ tilt, description was presented by Lindh et al. (8).
Elemental Analysis of Individual Cells by PIXE The elemental analysis was performed in the Studsvik Nuclear Microprobe. The analytical technique opted for this task was particleinduced X-ray emission (PIXE). The PIXE is a technique with multielem e n t capacity, permitting the determination of elements with Z > 10, provided the concentration of the element is > 0.5 ~g/g. For excitation of the target, a microbeam (3 i~m) of 2.5 MeV protons from a Van de Graaff accelerator at Studsvik was used. The induced spectra of characteristic X-rays were collected by a multichannel analyzer for 300-1800 s and stored on tape for later treatment (9). A technical account of the analytical instrument, including the latest developments, has been published elsewhere (10). The detection limit of the examined elements is 0.5 i~g/g or better. The precision of analysis was estimated as 5% and the accuracy 10-15%, as assessed by broad beam measurements. The actual accuracy cannot be estimated because of the lack of microstandards. By measuring the bremsstrahlung, which is proportional to the mass under the probe, a relative value (concentration) can be obtained for comparison of samples under the same conditions.
RESULTS AND DISCUSSION Elemental Profiles of the Erythrocytes Figure 1 shows the concentrations of selenium, calcium, magnesium, and zinc as a function of time. All concentration data are given as median values, dry weight. The dose of l-selenomethionine was rather Biological Trace Element Research
Vol. 12, 1987
104
Johansson and Lindh ERYTHROCYTES
0 ~Pg g-'
0
Pg g-,
20-
..........
0 ....
-Q
,o"
~""
/ / Mg
Q,
/
/
-
so
,
"6
10-
940
8-
. 69 2O
42-
0.5
o
O
o
,
1
2
I
I
;
4
;
;
7
8
I i
110
I
I
1'1
1'2 Months
Fig. 1. The concentrations of several elements in the erythrocytes (n = 20) are given as median values, dry weight. The filled bars represent selenium; A, Ca; o, Mg; and [E, Zn. The concentration of Mg should be read on the righthand scale. Unfilled symbols on the horizontal axis represent undetectable concentrations (
Interactions of Selenium with Metal Ions at the Cellular Level ERLAND JOHANSSON* AND ULF LINDH
Department of Physical Biology, Gustaf Wemer Institute, Uppsala University, Box 531, S- 751 21 Uppsala, Sweden ABSTRACT In a supplementation study in which organic selenium as l-selenomethionine was administered in low doses during 1 yr, alterations in the concentrations of metal ions in the erythrocytes and the neutrophil granulocytes were observed. In the erythrocytes, altered concentrations of zinc were parallel with selenium. The concentrations of magnesium, calcium, manganese, copper, and sulfur were not significantly altered. However, altered concentrations of iron and zinc were observed in the neutrophils. The concentrations of magnesium, calcium, manganese, copper, and sulfur were not significantly altered. The accumulation of selenium in individual blood cells was different from that obtained with supplementation of inorganic selenium. When organic selenium was supplemented, the thrombocytes accumulated more selenium than the erythrocytes and the neutrophil granulocytes. The observations indicate that selenium interacts with metal ions at the cellular level when supplemented in low doses. The chemical form of selenium might be important in nutrition and therapy in view of the interaction and distribution pattern at the cellular level. Index Entries" l-fielenomethionine; selenium; iron; zinc, calcium; trace elements; erythrocytes; thrombocytes; neutrophil granulocytes; metal ion interactions; nuclear microprobe; micro-PIXE.
INTRODUCTION The bioavailability of an element is a complex function of several factors. Among those are the chemical form of the element, uptake and *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1 01
VoL 12, 1987
102
Johansson and Lindh
transport kinetics, metabolic transformations, and genetic influence. There is no satisfactory model for the availability of trace elements accounting for interactions with other elements. To completely understand the mechanisms of bioavailability and the importance of interactions with other elements, it is essential to know the chemical form of the nutrient. Lack of analytical methods to assess the redox state and to determine the low concentrations has probably hampered the inclusion of the chemical form in the recommendations of dietary intake (RDA) of macro- and trace elements. It is therefore desirable to design experiments using trace elements in known chemical forms to cast light upon the concept of bioavailability. Using known chemical forms of trace elements facilitates identifications of interactions at the cellular level. This is one of the prerequisites for more precise models to assess the bioavailability of trace elements in humans. Selenium attracted an increasing amount of interest after the discovery of Keshan Disease (1,2) and Kashin Beck Disease (3), which appear to be the first documented human diseases with low selenium status as one important risk factor. In these studies, plasma selenium, whole blood selenium, and hair selenium were used to assess the selenium status. Blood cells with different life spans, however, might be more useful as a model (4) of bioavailability because the uptake and distribution of trace elements as well as interactions with other elements can be monitored using the nuclear microprobe (5). In addition, it is necessary to investigate the correlation between cell functions and trace element concentrations. Studies of the interactions of selenium with other elements at the cellular level are scarce. Studies by Lindh and Johansson (5), Johansson et al. (6), and Johansson and Lindh (7) showed that when inorganic selenium was supplied for I yr, interactions with other elements occurred at the cellular level, as reflected in the elemental profile of individual blood cells. When the selenium concentration was altered, some metal ions were altered in a regular manner. The concentrations of iron, copper, manganese, and zinc were altered in the neutrophil granulocytes, and the thrombocytes displayed altered concentrations of iron and zinc. Since l-selenomethionine is one form of selenium in food, e.g., wheat, we have supplied 1-selenomethionine in low doses to mimic physiological conditions. Early, and also long-term, effects of the distribution of an essential element and the interaction with other elements might be important when plain food is fortified with such an element.
MATERIAL AND METHODS Organic selenium (50 p~g Se/d) as l-selenomethionine was supplied per os (orally) to one volunteer (52 yr, 74 kg), who was apparently healthy and not subjected to medication, with one exception. During 10 Biological Trace Element Research
VoL 12, 1987
Interactions of Selenium with Metal Ions
103
d, from d 100 to 110, penicillin [3 tablets Fenoxypen (666 rag) 3 times a day] was administered to cure an acute infection. The study was completed in 1 yr. The concentration of selenium in Swedish plain food is low; the daily intake is estimated as 30 ~g selenium/d. Fish and liver was restricted to once or less a week.
Blood Sampling and Cell Separation Five milliliters of venus blood (EDTA anticoagulant) were drawn at the same time of day, 11 AM. The different types of blood cellwhich rated immediately after sampling. The blood cells were fractionated by centrifugation at different speeds. The erythrocytes were isolated at 150g, the neutrophils at 400g, and the thrombocytes at 1000g. After the separation the pellets were s u s p e n d e d in 0.32M sucrose before disen, ing on backings of thin Formvar film. The cells were then quench frozled freeze-dried, and stored in a desiccator before analysis. A more detai KV, 15~ tilt, description was presented by Lindh et al. (8).
Elemental Analysis of Individual Cells by PIXE The elemental analysis was performed in the Studsvik Nuclear Microprobe. The analytical technique opted for this task was particleinduced X-ray emission (PIXE). The PIXE is a technique with multielem e n t capacity, permitting the determination of elements with Z > 10, provided the concentration of the element is > 0.5 ~g/g. For excitation of the target, a microbeam (3 i~m) of 2.5 MeV protons from a Van de Graaff accelerator at Studsvik was used. The induced spectra of characteristic X-rays were collected by a multichannel analyzer for 300-1800 s and stored on tape for later treatment (9). A technical account of the analytical instrument, including the latest developments, has been published elsewhere (10). The detection limit of the examined elements is 0.5 i~g/g or better. The precision of analysis was estimated as 5% and the accuracy 10-15%, as assessed by broad beam measurements. The actual accuracy cannot be estimated because of the lack of microstandards. By measuring the bremsstrahlung, which is proportional to the mass under the probe, a relative value (concentration) can be obtained for comparison of samples under the same conditions.
RESULTS AND DISCUSSION Elemental Profiles of the Erythrocytes Figure 1 shows the concentrations of selenium, calcium, magnesium, and zinc as a function of time. All concentration data are given as median values, dry weight. The dose of l-selenomethionine was rather Biological Trace Element Research
Vol. 12, 1987
104
Johansson and Lindh ERYTHROCYTES
0 ~Pg g-'
0
Pg g-,
20-
..........
0 ....
-Q
,o"
~""
/ / Mg
Q,
/
/
-
so
,
"6
10-
940
8-
. 69 2O
42-
0.5
o
O
o
,
1
2
I
I
;
4
;
;
7
8
I i
110
I
I
1'1
1'2 Months
Fig. 1. The concentrations of several elements in the erythrocytes (n = 20) are given as median values, dry weight. The filled bars represent selenium; A, Ca; o, Mg; and [E, Zn. The concentration of Mg should be read on the righthand scale. Unfilled symbols on the horizontal axis represent undetectable concentrations (
