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,3
* Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706, and
Department of Nutritional Sciences, University of Missouri, Columbia, MO 65211
3To whom correspondence should be addressed. E-mail: Sunde{at}nutrisci.wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • biomarkers • glutathione peroxidase • molecular biology • RDA • requirements
The discovery that glutathione peroxidase-1 (GPX1,4 glutathione:H2O2 oxidoreductase, EC 1.11.1.9) contains Se (1) provided a biochemical marker that was important in setting animal and human Se requirements. The 2000 U.S. Recommended Dietary Allowances (RDAs) calculated an Estimated Average Requirement of 45 µg of Se that would maximize plasma GPX activity based on independent analysis of the data from studies in China (2) and New Zealand (3); after adjustment for variation, this resulted in the RDA of 55 µg of Se for both men and women (4). In an alternative approach, the WHO estimated the dietary Se intake necessary to achieve two-thirds of maximal plasma GPX activity, plus 16% interindividual variation, resulting in a Normative Requirement of 40 µg of Se for men and 30 µg Se for women (5). This diversity in approach and application when setting human Se requirements illustrates why further research is warranted on Se biomarkers and on the relation of biomarkers to health (4).
In the intervening years since the discovery that GPX1 is a selenoenzyme, advances in our understanding of the biochemistry and molecular biology of Se and its selenoproteins revealed why the GPXs and other selenoproteins are good biochemical markers for establishing Se status and Se requirements [for recent reviews, see (6,7)]. The early studies showing the effect of long-term Se depletion and supplementation on GPX illustrated that erythrocyte GPX activity and liver GPX activity are strongly correlated with dietary Se level up to the point at which Se no longer regulates the expression of these activities (8). The subsequent discovery that Se status also regulates GPX1 mRNA levels (9) led to a series of studies on the dose response of not only selenoprotein level but also selenoprotein mRNA level, showing that molecular biology markers could also be used to evaluate requirements (10–13). These studies also showed that the dramatic changes in liver GPX activity occur not only because Se is required for enzymatic activity but also because the GPX1 mRNA levels are also highly regulated by Se status. At least in rats, regulation of GPX1 mRNA by Se status is much more substantial than for any other selenoprotein mRNA, falling to as low as 6–10% of Se-adequate levels (9,10,14,15). Differences in the extent of decrease of activity and mRNA level in Se deficiency, and the point at which a plateau is first reached in the dietary Se response curve can explain the hierarchy of selenoprotein expression as animals go from adequate to deficient levels (15). These animal studies showed that there are profound homeostatic regulatory mechanisms that control the level of these selenoproteins, and clearly illustrate why certain selenoprotein levels are good biomarkers of Se status.
The advent of rapid molecular assays such as real-time PCR and microarrays suggests that these tools will soon be used for evaluating health and disease parameters in humans. Thus, developing tools for assessing Se status in humans using molecular biology markers seems an important area for study today. Liver selenoprotein mRNA levels would be a very effective tool for assessing human Se status, but are also highly invasive and thus impractical. To better characterize potential tissues for using molecular biology to assess Se status, we conducted the experiments here to begin to study the efficacy of using mRNA in whole blood for assessing Se status. We used ribonuclease protection analysis (RPA) to evaluate mRNA expression in whole blood and 16 other tissues of rats. These studies illustrate that rat blood can readily provide sufficient total RNA for molecular analysis, and that the level of GPX1 expression in blood ranks with major tissues that have high levels of GPX1 expression. Importantly, the levels of expression in whole blood reflect the levels in red cells rather than in white cells, further suggesting that whole blood mRNA analysis may be a convenient and effective biomarker worthy of further evaluation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In Expt. 2, male weanling rats, 21-d old, were fed a Se-adequate (0.2 µg Se/g as Na2SeO3) crystalline amino acid diet (10) that included 9.6 g/kg L-methionine (U.S. Biochemical) and 150 g/kg of all-rac-
-tocopheryl acetate to allow adequate growth and to prevent liver necrosis, respectively. After 28 d, the rats weighed 267 ± 4 g. The rats (n = 3) were anesthetized with ether, exsanguinated by cardiac puncture using heparinized syringes, and 16 tissues were removed and quick-frozen in liquid nitrogen for later RNA analysis. GPX activity was determined for red cells, plasma, and liver supernatants by the coupled assay procedure using 120 µmol/L H2O2; thus, only the Se-dependent GPX activity was measured (16). GPX4 activity was measured using 78 µmol/L phosphatidylcholine hydroperoxide as described previously (10). For each assay, an enzyme unit is defined as the amount of enzyme that oxidizes 1 µmol GSH/min under the specified conditions. Protein concentration was determined by the Lowry method (17). For whole blood, the red cell, and white cell fractions, total RNA was isolated by rapidly mixing 3 mL with Tri Reagent BD (Molecular Research Center) (3.75 mL/mL blood or blood fraction following manufacturer’s protocol) and frozen for later analysis. For rat tissues, total RNA was isolated from 
0.3-g portions of tissue by homogenization in guanidine isothiocyanate buffer followed by centrifugation (176,000 x g, 20 h) on 5.7 mol/L CsCl as described previously (9). Adrenal glands and olfactory bulbs were each pooled (pooled weights < 200 mg) for RNA analysis, and those samples analyzed with each set of tissue total RNAs. The RNA pellets were dissolved in diethyl pyrocarbonate-treated water and quantitated spectrophotometrically by A260 (
= 25 mL · mg–1 · cm –1).
In Expt. 3, male weanling rats (n = 3/group) were fed a basal Se-deficient 30% torula yeast diet (18), containing 0.007 µg Se/g diet by analysis and supplemented with 100 mg/kg all-rac--tocopheryl acetate, or the basal diet supplemented with 0.2 µg Se/g diet as Na2SeO3. After 28 d, blood and liver were obtained and analyzed for selenoenzyme activity and mRNA levels as described for Expt. 2.
The RPA was conducted as previously described (14,19). DNA probe templates were subcloned into the pBluescript-II (SK-) vector (Stratagene). In vitro transcription of antisense RNA probes was performed essentially according to the manufacturer’s protocol (Promega). Probes5 for GPX1, GPX4, TRR1, GPX3, SelP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized using 10–70 µCi of [-32P] UTP, purified by electrophoresis in a 6% polyacrylamide gel, eluted in 2 mol/L ammonium acetate containing 1% SDS, and ethanol precipitated for RPA. Total RNA (10 µg) was hybridized overnight at 45°C with a balanced mixture of the single-strand antisense RNA probes. Yeast tRNA served as a negative control. Each probe was also hybridized individually at twice the concentration using an individual probe with RNA from Se-adequate (0.2 µg Se/g diet) liver or kidney (for GPX3) to verify that the probe was in excess. Hybridization reactions were treated with RNase (40 mg/L RNase A, 2 mg/L RNase T1) for 45 min at 30°C. After RNase inactivation, the protected probe fragments were precipitated with ethanol and samples were analyzed in a 6% polyacrylamide gel. RNA protected from ribonuclease was visualized by autoradiography, and protected probe fragments in each sample were quantitated by direct imaging of the gel (Instant-Imager, Packard Instrument Company).
Data are presented as means ± SEM. One-way ANOVA, with differences between means assessed by Duncan’s multiple range analysis (20) (P < 0.05), was used to compare mRNA levels among different tissues. Analysis of tissue mRNA levels comparing Se-deficient and Se-adequate treatments was conducted using the unpaired Student’s t test (P < 0.05).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20 to 30/hpf. Thus, the white cell fraction was enriched as much as 250-fold in white blood cells relative to whole blood. In the enriched RBC fraction, the platelet estimation was 15 to 20/hpf and the WBC:RBC ratio could only be estimated at 1/500-1000 RBCs. In whole blood, the distribution of WBC was 43% neutrophils, 47% lymphocytes, 8% monocytes, and 2% eosinophils. WBC distribution in the white blood cell fraction was 29, 57, 10, and 4%, and estimated in the red cell fraction as 20, 72, 4 and 3%, respectively. Analysis of total RNA in the various blood fractions for GPX1, GPX4, TRR1, and GAPDH revealed that GPX1 was the major selenoprotein mRNA in red cells and in whole blood (Fig. 1). Similar levels of GPX4 were also present in red cells and in whole blood; this level was about half the level in the white cell fraction. TRR1 and GAPDH mRNAs were also observed in whole blood, but there were reduced levels in the red cell fraction relative to the white cell fraction. Interestingly, the GAPDH signal in white cells was very high, along with an elevated GPX1 level, whereas there was negligible GAPDH expression in whole blood and in red cells. Thus the low GAPDH signals in the red cell fraction and whole blood show clearly that white cells, with high levels of GAPDH mRNA, make a negligible contribution toward the mRNA levels detected in whole blood.
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5 times the level found in any other tissue. Blood was 6th in GPX4 mRNA abundance, with liver and kidney 3rd and 4th in ranking. For GPX3 expression, the kidney was highest as expected, because it is the major source of the circulating plasma GPX activity (21). Blood was 5th and liver was 6th, with testis having negligible GPX3 mRNA expression. For TRR1 mRNA expression, testis was high, with kidney and liver 3rd and 4th, and blood 7th in the hierarchy, respectively. Some of this TRR1 mRNA in whole blood likely arises from white cells, as shown in Figure 1. Last, SelP mRNA expression was also highest in testis and liver, followed by kidney; blood was 7th in the hierarchy. Although liver is thought to be the predominant source of circulating SelP levels (22), it is clear that other tissues including reticulocytes, testis, and kidney also have capacity to synthesize SelP. In contrast to high selenoprotein expression in blood, the expression of GAPDH in blood was very low relative to liver, testis, and kidney.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Unexpectedly, we found that substantial amounts of total RNA (>50 µg) could be obtained from 1 mL of whole blood. Sedimentation fractionation of whole blood into a white cell fraction and a red cell fraction revealed that the white cells contributed a small amount of the whole blood mRNA, based on the levels of GAPDH mRNA found in a white cell fraction vs. the red cell fraction or whole blood. Levels of GPX1 and GPX4 mRNA in whole blood and in the red cell fraction were very similar, further suggesting that whole blood total RNA might be an efficacious source of RNA to be used for assessing nutrient status.
This study is the first to report an evaluation of the gene expression pattern for a number of selenoproteins and across a comprehensive set of tissues. Comparison of GPX1 mRNA levels in total RNA obtained from whole blood and from 16 other rat tissues indicated that GPX1 mRNA levels in whole blood are similar to levels found in liver, kidney, and heart. As expected from previous studies (10,33), GPX4 mRNA levels were high in testis, but levels in blood were comparable to liver and kidney. Similarly, kidney had high levels of GPX3 mRNA (34), but levels in blood were comparable to heart and liver. TRR1 and SelP mRNA levels in blood were in a similar range but lower than in kidney and liver as well as testis, consistent with previous reports (13,22,35); broad tissue distribution of SelP expression may help explain the presence of SelP in brain and other tissues (36). Thus, these studies show that useful levels of total RNA can be readily obtained from whole blood and that the levels of expression, notably GPX1 mRNA levels, are comparable to levels found in the major organs that have been used to carefully assess nutrient status in animal models. In an initial experiment, we found that the fall in blood GPX1 mRNA levels was comparable to the fall in liver GPX1 mRNA, indicating that blood GPX1 mRNA at least in rats responds dramatically to Se deficiency.
Selenoenzyme activities across the tissues were not measured in this experiment. Correlation of the GPX1 mRNA levels determined here with GPX1 activities in 12 common tissues, determined in a similar study conducted previously in our laboratory with rats of the same strain and age and fed the same diet but with 0.1 µg Se/g diet (37), reveals that pancreas, cerebrum, testes, muscle, blood, adrenal glands, and blood all have a similar ratio of GPX1 activity to mRNA level, whereas thymus, heart, kidney, and lung all have proportionally more GPX1 activity than mRNA. A similar comparison of GPX4 mRNA levels with GPX4 activities (10,38) shows a nearly common ratio of GPX4 activity to mRNA in thymus, lung, heart, kidney, and testis but proportionally less (approximately half) GPX4 activity per mRNA in liver, cerebrum, and muscle. These differences may be due to altered rates of protein synthesis vs. protein degradation in these tissues, including differences in translational efficiency (14) or possibly due to the presence or absence of other peroxidases in these tissues, which contribute to measured activity.
On the basis of animal experiments, it is fairly clear that the most dramatic changes in the level of markers of Se status are found in liver and to some extent in plasma. When trying to use mRNA expression to assess Se status, however, use of liver or other internal organs is not practical for human studies, nor would sampling of the tissues of origin for plasma selenoproteins. Logical, less invasive sources of tissue for assessing nutrient status would include plasma, white cells, and whole blood, as well as cell scrapings from the mouth or other regions of the body. We found that 1 mL of whole rat blood would yield 50 µg of total RNA using a one-step denaturation process, thus showing that whole blood could be a major source of sufficient material that would permit multiple molecular biology assays; human blood can yield 15–20 µg of RNA/mL using this one-step isolation process (39).
Researchers have used white cells or platelets as a tissue for assessing Se nutrient status and nutrient requirements (23,24,40). Considerable material and manipulation, however, are required to obtain reproducible and significant amounts of platelet material, thus likely limiting the available total RNA for use in molecular biology analysis. Relative white cell and platelet numbers will also be affected by viral infection or other diseases such that this may introduce considerable variability. RBC, because of their long half-life of 17 wk in humans, offer advantages in addition to accessibility and large sample volume because the long lifespan might integrate nutrient status over a considerable period of time. In particular, this would mean that recent or transient nutrient supplementation or changes in dietary habits would not have large effects on the overall assessment of an individual’s nutrient intake. Several Se-repletion studies in humans showed that plasma GPX3 activity and platelet GPX can reach a plateau after 2–4 wk of Se supplementation, whereas RBC GPX activity requires 8–16 wk (23,24,31), showing that RBC offers the potential to integrate Se status over a longer period of time than do platelets or plasma. This could be a considerable advantage except for cases in which the study objective is to assess the effect of recent dietary changes or supplementation on nutrient status. There are several limitations on the use of blood RNA as well, including the effect of blood loss, e.g., menstruation, or disease, which might adversely influence the apparent nutrient status relative to the real nutrient status of an individual. Similarly, other nutrient deficiencies might cause microcytic or macrocytic anemias, which might adversely affect these parameters. At this point it is not clear whether RBC would be more affected than other tissues by changes in the status of other nutrients. An effective internal biomarker in red cell RNA would be helpful as a normalization standard in nutrient status assessment.
In summary, these initial studies showed that whole blood total RNA is readily available at least in rodents and has sufficient mRNA levels for GPX1 and GPX4, as well as perhaps some of the other selenoenzymes, such that whole blood is a potential source of RNA for use in molecular biology assessment of nutrient status. Additional studies in animals, including assessment of the effect of changes in blood loss and changes in infection, will be required to further demonstrate the usefulness of whole blood RNA for assessing nutrient status. Of course, similar studies will be necessary in humans before routine application of molecular biology approaches can be employed for assessing Se status and requirement as well as for other nutrients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by USDA 98–35200-6051.
4 Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX, glutathione peroxidase; hpf, high power field; RDA, Recommended Dietary Allowance; RPA, ribonuclease protection assay; SelP, selenoprotein-P; TRR1, thioredoxin reductase-1; WBC, white blood cell.
5 RPA probes were: GPX1 (X07365, bases 411–957), GPX4 (NM 017165, bases 209–653), TRR1 (NM031614, bases 209–599), GPX3 (NM022525, bases 430–810), SelP (NM019192, bases 987-1252) and GAPDH (NM017008, bases 1148–1463).
Manuscript received 14 May 2004. Initial review completed 10 June 2004. Revision accepted 15 July 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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