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Article

Metallothionein mRNA in Monocytes and Peripheral Blood Mononuclear Cells and in Cells from Dried Blood Spots Increases after Zinc Supplementation of Men¹

Center for Nutritional Sciences, Food Science and Human Nutrition Department, University of Florida, Gainesville FL 32611-0370

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A specific, sensitive and reliable index for assessment of human zinc status has not been developed, and continues to present a considerable challenge for nutritionists in the trace element field. We have focused on metallothionein (MT) expression as a potential index. A protocol involving 16 men and a 10-d supplementation period plus a 4-d postsupplementation period was used to examine the relative response of MT expression in erythrocytes, monocytes, peripheral blood mononuclear cells (PBMC) and cells from a dried blood spot (DBS). Zinc was supplemented at the current adult male recommended dietary allowance (RDA) of 15 mg. Erythrocyte MT protein, as measured by ELISA, increased gradually to about twofold over the placebo group during zinc supplementation and remained elevated for 4 d postsupplementation. Competitive reverse transcriptase-polymerase chain reaction showed that MT mRNA levels in both monocytes and PBMC increased (up to 4.7- and 2.7-fold, respectively) after 2 d of supplementation, with greater expression in monocytes compared with PBMC. Total RNA extracted from dried blood spots, representing cells from 50 µL of blood, showed a comparable change in MT mRNA upon zinc supplementation. In each leukocyte population isolated, when zinc supplementation was withdrawn, MT mRNA levels decreased. Collectively, these experiments show that, in men, MT gene expression increases during supplementation at the RDA, and that the DBS sampling method will be of value in measuring MT expression in a variety of clinical and survey situations.

KEY WORDS: • zinc • metallothionein • monocytes • lymphocytes • gene expression • humans

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Zinc has been known to be an essential nutrient for humans for many years and is required for many biochemical processes (Cousins 1996 ). Indicators of zinc status such as hair, urine, sweat, tears and saliva zinc (King 1990 , Van Wouwe 1995 , Van Wouwe et al. 1986 ) are not reliable. The plasma zinc concentration may be useful under some circumstances, but it is regulated homeostatically within a narrow range and thus adapts to a range of zinc intake levels. Similarly, activities of zinc metalloenzymes, including alkaline phosphatase, 5-nucleotidase, erythrocyte and plasma superoxide dismutase and several others, have had mixed success as indices of zinc status (Cousins 1996 ).

A specific, sensitive and reliable indicator for human zinc status is still lacking. Because marginal zinc deficiency is thought to be common, especially in developing countries, such a method is of considerable interest (Hambidge 2000 ). This has produced a strong incentive for developing assays of status that would be applicable to experimental and clinical studies and for survey research.

Metallothionein (MT)³ is a small, cysteine-rich, metal-binding protein that can bind zinc and other heavy metals with high affinity; it has no defined function, although many have been advanced, most with considerable experimental evidence (Davis and Cousins 2000 ). Metallothionein expression has been related positively to dietary zinc intake in numerous studies with animals (Cousins and Lee-Ambrose 1992 , Sato et al. 1984 ) and humans (Grider et al. 1990 , Sullivan et al. 1998 , Thomas et al. 1992 ).

To explore MT expression as a potential index for human zinc status, we first developed a competitive ELISA for measuring human erythrocyte MT protein concentrations (Grider et al. 1989 ) and subsequently used this technique in human studies (Grider et al. 1990 , Thomas et al. 1992 ). In an ongoing effort, this method was later optimized to a more practical sandwich ELISA (Sullivan et al. 1998 ). We also developed a sensitive alternative method, competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR), to quantify MT mRNA in monocytes (Sullivan and Cousins 1997 , Sullivan et al. 1998 ). In those studies with monocytes, metallothionein protein was found to increase about twofold, whereas MT mRNA levels increased by more than fivefold upon supplementation with zinc at 50 mg/d.

Although MT mRNA in monocytes is quite sensitive to zinc supplementation, the proportion of monocytes in peripheral blood is small, and the isolation procedure is relatively long. In contrast, peripheral blood mononuclear cells (PBMC) are more abundant than monocytes, and their separation by density gradient centrifugation is less time-consuming. Consequently, it seemed appropriate to examine MT mRNA levels in PBMC, as an alternative to monocytes, after zinc supplementation.

Dried blood spot (DBS) absorbent paper has been used for body fluid sample collection and analysis for a long time, and is now widely used for sample collection of forensic, pediatric and epidemiologic samples. The DBS sample collection approach has been applied successfully to human nutritional assessment studies for amino acids (Becker et al. 1985 ), lipids (Hirst and Beswick 1993 ) and, more recently, folate (O’Broin and Gunter, 1999 ). No report describing the use of the DBS for zinc status assessment in humans is available. With the newly developed sensitive molecular biological methods, such as C-RT-PCR, it may be possible to detect MT mRNA levels in samples as small as those produced with the DBS technique.

The objectives of the present experiments were as follows: 1) to compare erythrocyte MT protein and monocyte MT mRNA changes after zinc supplementation at the current adult male recommended dietary allowance (RDA) of 15 mg/d (NRC 1989 ) with the 50 mg/d used previously (Sullivan and Cousins 1997 , Sullivan et al. 1998 ); 2) to compare the response of metallothionein mRNA levels in PBMC with those in monocytes after zinc supplementation; 3) to evaluate erythrocyte MT protein changes produced by zinc supplementation at 15 mg/d; and 4) to explore the feasibility of measuring MT mRNA levels in DBS samples obtained from control and zinc-supplemented human subjects.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human subjects.

The study was approved by the University of Florida Institutional Review Board, and informed consent was obtained from all subjects. Free-living healthy men between the ages of 19 and 31 y were chosen for this study. All subjects were nonsmokers with no history of chronic illness, no recent surgery and no routine use of medications or drugs. Two weeks before the experiment started, subjects were advised to eat a balanced diet with foods selected from each food group, avoiding zinc-rich foods such as oysters, to refrain from alcohol consumption and to stop any nutritional supplements. Initial and postsupplementation blood chemistry profiles were performed (SmithKline Beecham, Collegeville, PA), and subjects whose profiles fell out of any normal range were excluded from the study. The profile included total cholesterol, HDL cholesterol, glucose, urea nitrogen, creatinine, Na, K, Cl, Ca, total protein, albumin, globulin, bilirubin, alkaline phosphatase and aspartate aminotransferase.

Experimental protocol.

The subjects (n = 16) were divided randomly into zinc-supplemented (treatment) or placebo control groups of equal size. The supplement was produced by mixing 1 volume of zinc sulfate (analytical grade; Mallinckrodt, Paris, KY) with 3 volumes of sucrose, followed by thorough grinding and mixing. Samples of the zinc sulfate/sucrose mixture were measured for zinc content by air acetylene flame atomic absorption spectrophotometry (AAS) to confirm homogeneity of the sample dose, and then placed in gelatin capsules. Subjects in the supplemented group were given 15 mg zinc (as this mixture), whereas control subjects received an equivalent amount of sucrose as the placebo. This supplemental level is the current RDA for adult males (NRC 1989 ). The study was divided into two phases, i.e., 10 d of zinc supplementation and 4 d of postsupplementation. Compliance was verified by consumption of the supplement in the presence of the investigator before each morning meal.

One tube of venous blood (7 mL) was withdrawn into trace element–free tubes (Becton Dickinson Vacutainer No. 6457; Fisher Scientific, Pittsburgh, PA) on d 0, 2, 4, 6, 8, 10, 12 and 14. All venous blood samples were drawn between 0800 and 0900 h, after an overnight fast and before the morning meal. At the end of the supplementation period, a blood sample (7 mL) was drawn for HDL cholesterol and hemogram differential analyses to investigate potential effects of supplementation on these parameters (Table 1 ). All blood samples were processed at room temperature immediately.

To isolate monocytes, whole blood (3 mL) was mixed (10:1) with Dextran 500 (60 g/L) in 154 mmol/L NaCl by inversion and allowed to stand for 40 min to separate plasma. The top layer (leukocyte-rich plasma) and buffy coat were removed, and monocytes were isolated using NycoPrep 1.068 (Life Technologies, Rockville, MD) to a purity of 80% as described previously (Sullivan and Cousins 1997 ). To isolate PBMC, another 3 mL of whole blood was carefully layered onto 3 mL of Histopaque 1.077 (Sigma Diagnostics, St. Louis, MO) and centrifuged at 400 x g for 30 min. The interface containing mononuclear cells was removed and the cells were washed twice with PBS solution and centrifuged at 250 x g for 10 min. The erythrocytes from the monocyte and PBMC isolations were combined and washed with ice-cold 154 mmol/L NaCl and lysed by addition of ice-cold MilliQ H₂O as described earlier (Thomas et al. 1992 ).

Sandwich ELISA for measurement of erythrocyte lysate metallothionein.

Erythrocyte MT concentrations were measured by a sandwich ELISA using monoclonal anti-human metallothionein (hMT)-1 and -2 and chicken anti-hMT antibodies modified from the method developed previously (Sullivan et al. 1998 ). Antibodies were diluted in blocking buffer (1X PBS, 3% bovine serum albumin, 0.02% NaN₃) unless otherwise noted. Monoclonal anti-hMT antibody was diluted to 0.5 mg/L in 10 mmol/L sodium acetate buffer, pH 5.5, and activated with NaIO₄. Carbohydrate binding plates (Costar, Cambridge, MA) were coated with 100 µL/well of activated antibody and incubated for 1 h. Nonspecific sites were then blocked with SuperBlock buffer (Pierce, Rockford, IL) in PBS according to manufacturer’s instructions. Purified hMT-1 (Sullivan et al. 1998 ), serially diluted in 1X PBS (pH 7.4) or erythrocyte lysate, was added and incubated for 2 h; then the plates were washed with washing buffer (PBS, pH 7.4; 4 mmol/L Tween 20) three times. Chicken anti-hMT immunoglobulin (Ig)Y was added and incubated for 2 h. The plates were washed three times and rabbit anti-chicken IgG alkaline phosphatase conjugate (Sigma Chemical, St. Louis, MO) was added (diluted 1:3000) and incubated for 1 h. Plates were washed three times and p-nitrophenyl phosphate substrate (Pierce) was added. After an incubation period, absorbance was measured at 405 nm and MT concentrations were calculated by linear regression.

RNA isolation and competitive RT-PCR for determination of metallothionein mRNA levels in isolated human cells.

Total RNA from purified PBMC and monocytes from each subject was extracted using TRIzol reagent (Life Technologies) as described previously (Sullivan et al. 1998 ). Concentration and purity of the RNA were determined by measuring the absorbance in TE buffer (10 mmol/L Tris-HCl, pH 8.0; and 1 mmol/L EDTA) at 230, 260 and 280 nm.

The level of MT mRNA was determined by a modified C-RT-PCR described previously (Sullivan and Cousins 1997 ). In brief, total RNA from cells was diluted to 0.1 g/L and denatured for 5 min at 70°C. Reverse transcription reactions were performed with the following components: 0.2 µg total RNA, 200 mmol/L dithiothreitol, 0.4 µmol/L mixed dNTPs, 50 µmol/L d(pT)T₁₂ primer, and 300 U reverse transcriptase in a 20-µL reaction volume for 60 min at 37°C, followed by heat inactivation for 5 min at 95°C (Ausubel et al. 1995 ). A previously developed 180-bp cDNA competitor was used to calculate the concentration of the target MT cDNA (Sullivan and Cousins 1997 ). The following primers were used to simultaneously amplify both the 180-bp competitor cDNA and the 200-bp target MT cDNA templates: 5' primer, 5' ATG GAT CCC AAC TGC TCC TGC G 3'; and 3' primer, 5' AGG GCT GTC CCA ACA TCA GGC 3'. In these experiments, twofold dilutions of the competitor cDNA template were added to a constant amount of the target MT cDNA. Both competitor and target cDNA were coamplified using a PCR protocol with 30 cycles of denaturation, annealing and extension at 95, 55 and 72°C for 30 s, 1 min and 1 min, respectively. In these experiments, 2 µL of the above RT reaction and 2 µL of appropriate competitor stock were used in a 20-µL PCR reaction. The RT-PCR products were separated on an 8% polyacrylamide gel at 40 mV and stained with ethidium bromide (Ausubel et al. 1995 ). The gels were then photographed, and the densities of bands were analyzed by Intelligent Quantifier software (Bio Image, Ann Arbor, MI). The MT mRNA concentration of the sample was assumed equal to the known concentration of competitor cDNA that gave a 1:1 signal ratio with the target MT cDNA (Fig. 1 ) and did not account for the 21-bp difference in size between target and competitor.

View larger version (31K): [in this window] [in a new window]   Figure 1. Representative competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR) assay to measure metallothionein mRNA in total RNA from human blood mononuclear cells. (A) Twofold dilutions of a known amount of competitor cDNA were mixed with cDNA generated by reverse transcription from mononuclear cell RNA, and both were amplified by PCR. Total RNA (0.2 µg) was reverse transcribed in a 20-µL reaction. The PCR reaction (20 µL) contained 2 µL of the above reaction (0.02 µg RNA) and 2 µL of a competitor cDNA with a stock concentration of 0.023–1.472 amol/µL. The products were resolved on an 8% polyacrylamide gel, stained with ethidium bromide and photographed under UV light for densitometry. (B) Relative band densities of the 180-bp competitor cDNA to the 201-bp human metallothionein (hMT) cDNA generated by PCR. The point at which lines intersect is the point at which a 1:1 relationship exists and the concentration of the competitor cDNA band equals that of the hMT cDNA band.

Aliquots of venous whole blood (50 µL) obtained from each subject as above were spotted within preprinted circles onto sterile S&S 903 specimen collection cards (Schleicher & Schuell, Keene, NH; lot no. W961A01520). All DBS cards were suspended horizontally while spotting to prevent any blood from going through, and then placed on a clean paper towel to air dry. To determine MT mRNA stability at different conditions, the DBS collection cards were air-dried for different lengths of time at room temperature, then stored at either 4°C or -20°C with or without being sealed in plastic heat-sealed bags.

MT mRNA levels in DBS samples were measured in basically the same manner as for PBMC and monocytes, but with several modifications. Dried blood spots were cut out and placed in a sterile tube with 1 mL of TRIzol reagent, and RNA was extracted as above. After the RNA pellet received the last wash with 70% ethanol, appropriate amounts of autoclaved MilliQ H₂O were added to the dried RNA pellet without measurement of the RNA concentration. Reverse transcription was carried out immediately. Initially, blank paper spots of the same size were used as controls to confirm that there was no measurable MT mRNA activity. Similarly, replicate spots of the same sample were tested to ensure that factors such as RNase activity were not influencing the results.

Determination of plasma zinc and erythrocyte lysate zinc and protein concentrations.

Venous whole blood (1 mL) was centrifuged at 1500 x g for 10 min. Plasma and erythrocyte lysate were diluted 1:5 and 1:14, respectively, with distilled deionized MilliQ H₂O. Zinc concentration was measured using air acetylene flame AAS. Protein concentrations for erythrocyte lysate, MT and antibody were measured by the method of Lowry et al. (1951) with bovine serum albumin as the standard.

Statistical analysis.

Data were analyzed using Statistical Analysis System software (Windows version 6.12; SAS Institute, Cary, NC). Repeated-measures ANOVA was used to test the effect of the zinc supplementation on within-subject variation and the interaction of within-subject variation and zinc supplementation on plasma and erythrocyte zinc concentrations, as well as MT mRNA levels in PBMC, monocytes and DBS during the experimental period. Treatment means were compared using a least-square means (LSMEANS) statement with printed all possible probability value option (SAS Institute 1988 ). Values are means ± SEM

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Blood chemistry.

Prescreening data showed that all subjects participating in this study had normal blood chemistry, i.e., they were within the range of normal values (data not shown). Before the start of the treatments, HDL cholesterol levels in subjects that comprised the control and zinc supplemented groups were 1.3 ± 0.1 and 1.1 ± 0.1 mmol/L, respectively. These values are not different (P > 0.05). Supplementation with zinc for 10 d at 15 mg/d did not affect (P > 0.05) any of these values, including HDL levels and white blood cell differential counts (Table 1) .

Plasma and erythrocyte zinc concentrations.

Plasma zinc concentrations for subjects receiving zinc supplements were higher than those in the control group (P < 0.001) (Fig. 2A ). There were no differences in basal plasma zinc concentrations between the groups at d 0. During zinc supplementation, these concentrations increased up to 17.5 µmol/L for the supplemented group at 2 d. After d 2, plasma zinc levels for this group started to decrease gradually. Significant differences (P < 0.001) were found between the placebo and zinc-supplemented groups only at 2, 4 and 6 d. Plasma zinc concentrations for the control group remained unchanged (P > 0.05) throughout the study.

View larger version (21K): [in this window] [in a new window]   Figure 2. Plasma and erythrocyte lysate zinc concentrations in control and zinc-supplemented men. The subjects were given 15 mg Zn/d or a placebo for 10 d and no zinc supplementation for an additional 4 d. (A) Plasma zinc concentration. (B) Erythrocyte zinc concentration. Values are means ± SEM, n = 8. Mean is significantly different from corresponding mean of control subjects (***P < 0.001).

Erythrocyte metallothionein concentrations.

Overall, MT concentrations (Fig. 3 ) in erythrocytes were affected by zinc supplementation (P < 0.01). The erythrocyte MT concentrations at 0 d in the control and zinc-supplemented groups were similar (P > 0.05), 29 ± 2 and 26 ± 3 µg/g protein, respectively. After zinc supplementation was initiated, although erythrocyte MT concentrations increased gradually in the treatment group, whereas values for the control group remained unchanged, no significant differences (P > 0.05) were found before d 8 of supplementation. At d 10, MT concentrations for supplemented subjects reached their highest values (48 ± 8 µg/g protein) and were significantly higher (P < 0.01) than those of the control group (26 ± 1.2 µg/g protein). Erythrocyte MT concentrations started to decrease after zinc supplementation was stopped; however, values in the supplemented subjects were still higher than those in the placebo group at d 12 (P < 0.01; 39 ± 6 vs. 21 ± 1 µg/g protein, respectively) and d 14 (P < 0.05; 35 ± 5 vs. 24 ± 2 µg/g protein, respectively).

View larger version (22K): [in this window] [in a new window]   Figure 3. Metallothionein (MT) protein concentrations in erythrocytes from control and zinc-supplemented men. The subjects were given 15 mg Zn/d or a placebo for 10 d and no zinc supplementation for an additional 4 d. MT concentrations of the erythrocyte lysate were measured by sandwich ELISA and are expressed as µg MT/g protein. Values are means ± SEM, n = 8. Mean is significantly different from corresponding mean of control subjects (*P < 0.05; **P < 0.01).

Both monocytes and PBMC MT mRNA levels responded dramatically to zinc supplementation. Monocytes had higher MT mRNA levels than PBMC throughout zinc supplementation (Figs. 4 , 5 ). The levels in monocytes increased from 11.0 ± 1.7 to 43.0 ± 3.6 amol/µg RNA, whereas the levels in PBMC increased from 7.7 ± 0.5 to 20.0 ± 1.9 amol/µg RNA after zinc supplementation for 2 d. During further supplementation, monocyte MT mRNA levels increased up to 4.7-fold compared with the control group, whereas PBMC mRNA levels increased up to 2.7-fold. These levels were significantly (P < 0.001) higher in the zinc-supplemented subjects than those in the control group at each time point blood was collected. The higher levels were maintained until the end of supplementation. Both monocyte and PBMC MT mRNA levels in the treatment group decreased to levels near those in the control group (P > 0.05) 2 d after zinc supplementation was stopped (19.0 ± 1.4 vs. 13.5 ± 2.5 amol/µg total RNA for monocytes, 12.0 ± 1.7 vs. 9.2 ± 1.1 amol/µg total RNA for PBMC).

View larger version (25K): [in this window] [in a new window]   Figure 4. Metallothionein (MT) mRNA levels in purified monocytes from control and zinc-supplemented men. The subjects were given 15 mg Zn/d or a placebo for 10 d and no zinc supplementation for an additional 4 d. Monocytes were purified from venous blood by NycoPrep 1.068 gradient centrifugation and were the source of the total RNA used for reverse transcription. MT mRNA was measured by competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR) and expressed as amol MT mRNA/µg total RNA. Values are means ± SEM, n = 8. Mean is significantly different from corresponding mean of control subjects (***P < 0.001).

View larger version (24K): [in this window] [in a new window]   Figure 5. Metallothionein (MT) mRNA levels in purified peripheral blood mononuclear cells (PBMC) from control and zinc-supplemented men. The subjects were given 15 mg Zn/d or a placebo for 10 d and no zinc supplementation for an additional 4 d. PBMC were purified from venous blood by Histopaque 1.077 gradient centrifugation and were the source of the total RNA used for reverse transcription. MT mRNA was measured by competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR) and expressed as amol MT mRNA/µg total RNA. Values are means ± SEM, n = 8. Mean is significantly different from corresponding mean of control subjects (**P < 0.01; ***P < 0.001).

A number of pilot experiments (data not shown) were carried out to develop the DBS method described here. It was established that the DBS could be cut away from the remainder of the filter card and placed directly in Trizol reagent (20 min with occasional vortexing) to extract the RNA. Homogenization was not necessary. There was no MT mRNA found on blank filter cards. It was essential to air-dry the cards at room temperature for at least 2 h, but they could be kept at room temperature for up to 24 h with no loss of MT mRNA level. If the blood spots were not completely dry before storage, there was a loss of MT mRNA, presumably because RNase activity is related to the moisture content of the blood spot. Samples, once dried and stored in air-tight plastic bags, could be kept at -20°C for 1 mo or at 4°C for at least 10 d without showing significant loss of MT mRNA. MT mRNA levels obtained with the DBS were comparable to those from 50 µL of whole blood (data not shown). The interassay CV of the C-RT-PCR using the DBS as the source of the total RNA was 14% and the intra-assay CV was 8.6%.

Metallothionein mRNA levels in DBS responded to zinc supplementation in a manner similar to those in monocytes and PBMC (Fig. 6 ). Starting from d 2, DBS from zinc-supplemented subjects had a greater (P < 0.001) MT mRNA content compared with those from the placebo group (0.71 ± 0.22 vs. 0.16 ± 0.02 amol MT mRNA/DBS, respectively). Significantly higher levels of MT mRNA (P < 0.05) were still found 2 d postsupplementation (0.36 ± 0.05 vs. 0.19 ± 0.04 amol MT mRNA/DBS). There were no differences (P > 0.05) at d 14 between the two groups (0.25 ± 0.049 vs. 0.22 ± 0.02 amol/DBS).

View larger version (24K): [in this window] [in a new window]   Figure 6. Metallothionein (MT) mRNA levels in leukocytes from dried blood spot (DBS) samples from control and zinc-supplemented men. The subjects were given 15 mg Zn/d or a placebo for 10 d and no zinc supplementation for an additional 4 d. A dried blood spot comprising 50 µL of whole venous blood was the source of the total RNA used for reverse transcription. MT mRNA was measured by competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR) and expressed as amol MT mRNA/DBS. Values are means ± SEM, n = 8. Mean is significantly different from corresponding mean of control subjects (*P < 0.05; **P < 0.01; ***P < 0.001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Erythrocyte zinc concentrations in this study did not reflect zinc supplementation. Erythrocytes contain a large amount of zinc, chiefly as carbonic anhydrase (Thompson 1991 ). Therefore, recent changes in dietary zinc intake as in this study may not be sufficient to alter zinc levels in erythrocytes, which have a 120-d life span. This is in agreement with Neggers et al. (1997) , who suggested that short-term changes in dietary intake of zinc may not be reflected in zinc levels in erythrocytes because of the relatively long life span. Supplementation for longer periods of time, which would encompass more of the erythrocyte life span, might be expected to increase zinc concentrations of erythrocytes. However, our previous results (Thomas et al. 1992 ) and those of others (Davis et al. 2000 ) with supplementation at 50 mg/d did not result in such changes in studies of 30 and 90 d duration, respectively.

We have suggested that zinc in biology has the following three categories of function: catalytic, structural and regulatory (Cousins 1996 ). Each index of zinc status that has been investigated thus far can be placed within one of these functional categories. The catalytic roles played by zinc have received the most attention as indicators of reduced zinc status. This group includes the zinc metalloenzymes alkaline phosphatase, 5'-nucleotidase and peptidyl-dipeptidase. Results with any of these have been equivocal [reviewed in Cousins (1996) and King and Keen (1999) ]. Few studies have examined the effect of zinc supplementation on zinc-dependent enzymes. Recently, PBMC 5'-nucleotidase and plasma superoxide dismutase activities were increased during a 90-d supplementation with 50 mg Zn/d (Davis et al. 2000 ). Structural roles of zinc, e.g., zinc finger proteins (Klug and Schwabe 1995 ) or CD4/CD8 receptor complexation with the protein-tyrosine kinase p56^lck (Huse et al. 1998 ), as possible measures for status assessment have not been evaluated.

New potential biomarkers for assessing zinc status, based on the regulatory function of zinc (Cousins 1996 ), have been of increasing interest in the past decade. Evidence is increasing that zinc regulates the expression of specific genes (Cousins 1998 ). This could occur by the metal response element mode of transcriptional control (Andrews 2000 , Samson and Gedamu 1998 ) or through a zinc-dependent process that causes a cascade of events leading to a change in the cellular abundance of a specific protein through altered transcription/translation or post-translational modification (Blanchard and Cousins 2000 ). A variety of zinc-regulated genes, including MT and some zinc transporters, may serve as markers to allow the use of this functional modality (regulatory function) in status assessment. It is for this reason that we developed methods to evaluate changes in hMT gene expression.

Direct and indirect ELISA have been used successfully to measure MT protein in several studies in rat plasma (Akintola et al. 1995 ) and human erythrocyte lysate (Grider et al. 1989 and 1990 , Sullivan et al. 1998 , Thomas et al. 1992 ). Metallothionein protein levels in monocytes and PBMC have not been measured because, as pointed out by Sullivan et al. (1998) , even though MT levels in those cells are higher than in other leukocytes (Harley et al. 1989 , Pauwels et al. 1994 ), the abundance is too low to be measured by ELISA. Compared with the rapid increase in MT mRNA in monocytes, erythrocyte MT protein showed a significant increase in our subjects after 8 d of zinc supplementation. This is in agreement with other studies (Grider et al. 1990 , Sullivan et al. 1998 ). It is assumed that in most situations, protein levels are proportional to mRNA levels in cells. However, because erythrocytes are nonnucleated cells, the abundance of constituent proteins is a reflection of genes expressed in differentiating erythroid committed stem cells while in the bone marrow. As we hypothesized earlier, erythrocyte MT reaches a steady state of turnover in which synthesis programmed in the marrow as a function of both dietary zinc intake and zinc status is balanced against degradation in mature erythrocytes, which have a long life span (Huber and Cousins 1993a and 1993b ).

The zinc concentration in PBMC has been suggested as a reliable and sensitive indicator of zinc status because of its short average half-life (6 h) (Thompson 1991 ). Monocytes have the highest level of zinc among the various leukocytes (Goode et al. 1989 ). However, measuring zinc concentration of PBMC is difficult because a large amount of blood is required to isolate a sufficient number of cells to measure the zinc content. Alternatively, levels of specific mRNAs expressed in leukocytes that change in response to zinc supplementation and withdrawal provide a new opportunity to apply extremely sensitive technologies as indices of human zinc status.

Metallothionein expression in monocytes and lymphocytes in culture is induced by zinc (Mesna et al. 1990 , Steffensen et al. 1991 , Yamada and Koizumi 1991 ). We used C-RT-PCR to test the feasibility of measuring MT mRNA levels in cells derived from human subjects and to explore alterations in MT expression due to an increase in zinc status (Sullivan and Cousins 1997 , Sullivan et al. 1998 ). In the current experiments, monocyte MT mRNA levels were related positively to dietary zinc 2 d after supplementation started and remained elevated until zinc supplementation was stopped. These data coincide exactly with our previous results. Of particular interest is that, compared with a more than fivefold increase in monocyte MT mRNA levels when subjects received 50 mg Zn/d (Sullivan et al. 1998 ), our subjects supplemented with 15 mg Zn/d averaged a fourfold increase. This suggests that, at an intake of 50 mg Zn/d, MT expression in monocytes is near the maximal level possible.

It was within our expectation that MT mRNA levels in PBMC would be lower than those found in monocytes. Harley et al. (1989) found that absolute MT protein levels in monocytes were threefold higher than in lymphocytes in both basal and Cd²⁺-induced conditions. Peripheral blood mononuclear cells, separated in our study by Histopaque 1.077, contained both lymphocytes and monocytes. Normally, lymphocytes account for 20–40% of the total leukocyte population, whereas monocytes account for 2–8% (Ganong 1995 ). Therefore, the observation that MT mRNA levels in PBMC are 25% that found in monocytes is not surprising.

Leukocyte production increases with illness and infection (Ganong 1995 ). Zinc is known to play a central role in the immune system and involves proliferation and differentiation of many leukocyte populations (Fraker and King 1998 ). The synthesis of metallothionein is induced by many physiologic factors, including infection [reviewed in Andrews (2000) , Cousins (1996) , Davis and Cousins (2000) ]. The C-RT-PCR assay described here will not be affected by the number of cells produced because the approach is based on a finite amount of total cellular RNA (Sullivan et al. 1998 ). The blood chemistry profiles and white blood cell differentials in our two experimental groups were not different; therefore, in this study, the change in MT mRNA was related to zinc, not a difference in leukocyte cell types.

Metallothionein expression might be altered during infection. Most of the evidence for this is based on the response of the liver to infection and other stresses [reviewed in Cousins (1985) ]. Because hormone and cytokine regulation of MT is tissue specific (Davis and Cousins 2000 ), leukocytes may not respond to such stimuli. Therefore, to demonstrate that leukocyte MT mRNA is affected only by zinc intake in human subjects, a different set of experimental conditions and more experiments on MT expression under many physiologic conditions are required. This is particularly true in the case of the DBS approach, which is based on volume of blood rather than a total RNA measurement.

With sensitive techniques such as PCR and RT-PCR, blood samples spotted onto filter papers have been used successfully in newborn screening programs for the detection of gene mutations and HIV-1 RNA (Audrezet et al. 1993 , Cassol et al. 1997 , Fiscus et al. 1998 , Jinks et al. 1989 ). Some mRNAs can be detected with as few as 200 cells with RT-PCR (Alard et al. 1993 ). We reasoned that the measurement of MT mRNA levels in DBS with C-RT-PCR should not be a technical problem because there are 1.5 x 10⁵ leukocytes in a DBS, which represents 50 µL whole blood. Fiscus et al. (1998) found a substantial variation in quantitation of HIV-1 RNA with samples collected on Schleicher & Schuell No. 903 filter paper cards, but the results were very reproducible with newly available guanidinium isothiocyanate-treated (S&S Isocode) paper. The authors suggested that RNase contamination during sample processing might be a problem with Schleicher & Schuell No. 903 filter paper, but eliminated with guanidinium isothiocyanate-treated Isocode paper. In our study, all DBS cards used were from the same lot and, in our experience, reproducibility can be achieved by carefully handling papers and samples at all stages to avoid RNase contamination. Furthermore, the filter paper we used did not display appreciable endogenous RNase activity.

With the stability of MT mRNA on dried filter paper, the sensitivity and reproducibility of the C-RT-PCR method, and the convenience of the DBS sampling procedure, blood samples could be easily collected in the field from a finger- or heel-stick, dried on filter paper and sent to other locations for analysis. We believe that this cost-effective DBS method can provide an accurate measure of MT mRNA level and has potential application for both nutritional and clinical studies with humans in a variety of settings. Because the basal level of MT mRNA in the nonsupplemented subjects in this study was measurable, it is clear that these levels may decrease when dietary zinc intake/zinc status is reduced.

The response of MT gene expression to a daily zinc supplement of 15 mg/d has two interesting ramifications. The first is that zinc consumption, as a single supplement or as a combination vitamin/mineral supplement, is increasing. Data from NHANES III indicate that 16% of American adults are taking a supplement containing zinc (Moss et al. 1989 ). Although bioavailability varies greatly among zinc sources used in supplements, many provide at least 15 mg/d of highly bioavailable zinc. Zinc preparations for putative therapeutic purposes frequently recommend such doses more than once a day. As shown by our study, zinc supplementation at the current RDA has an effect at the genome level with respect to the MT gene. Moreover, the effect on gene regulation lasts as long as the zinc supplement is provided. The second is that, because the biological role of this metalloprotein has not been established [reviewed in Davis and Cousins (2000) ], the significance of MT induction by a zinc supplement is not clear. These results and those presented earlier (Sullivan et al. 1998 ) show that there is a detectable basal level of MT expression in erythrocytes and PBMC in subjects who are consuming a diet common to adult males in the U.S. This would lead to the notion that a basal level of MT production is required for normal cellular functions. Furthermore, a suggested role of MT in defense against reactive oxygen radicals or as a redox protein could lead to the interpretation that increased MT expression in cells, e.g., mononuclear cells, as shown in the present study, is beneficial and supplementation enhances such an effect. In contrast, the increase in expression during zinc supplementation could be viewed as a defensive action. Metal detoxification is one of the proposed functions of MT. If MT induction over basal levels of synthesis is a toxicologic response, supplementation with zinc, even at the current RDA, would have to be viewed with some caution.

In summary, we conclude from this study that MT mRNA levels in PBMC, as measured by C-RT-PCR, are as sensitive to zinc supplementation as MT mRNA in purified monocytes. Furthermore, zinc supplementation at 15 mg/d increases monocyte MT mRNA levels nearly as much as 50 mg/d. Finally, the DBS sampling method is amenable to the C-RT-PCR assay for hMT mRNA, and thus may be of value in measuring MT expression in which sample size is extremely limited.

	ACKNOWLEDGMENTS

 
We thank Jeff Bobo and Jerry Williamson for their help with the subjects in this and pilot studies, and Raymond K. Blanchard for helpful discussions on the C-RT-PCR methodology.

	FOOTNOTES

 
¹ Supported by Research Grant DK 31127 from the National Institute for Diabetes and Digestive and Kidney Diseases, and Boston Family Endowment Funds.

³ Abbreviations used: AAS, atomic absorption spectrophotometry; C-RT-PCR, competitive reverse transcriptase-polymerase chain reaction; DBS, dried blood spot; hMT, human metallothionein; Ig, immunoglobulin; MT, metallothionein; PBMC, peripheral blood mononuclear cells; RDA, recommended dietary allowance.

Manuscript received March 23, 2000. Initial review completed April 14, 2000. Revision accepted May 15, 2000.

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