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Clinical Trial
, 103 (6), 1699-704

Zinc Supplementation of Young Men Alters Metallothionein, Zinc Transporter, and Cytokine Gene Expression in Leukocyte Populations

Affiliations
Clinical Trial

Zinc Supplementation of Young Men Alters Metallothionein, Zinc Transporter, and Cytokine Gene Expression in Leukocyte Populations

Tolunay Beker Aydemir et al. Proc Natl Acad Sci U S A.

Abstract

An effective measure to assess zinc status of humans has remained elusive, in contrast to iron, where a number of indicators of metabolism/function are available. Using monocytes, T lymphocytes, and granulocytes isolated by magnetic sorting and dried blood spots (DBS) derived from 50 mul of peripheral blood, we evaluated the response of metallothionein (MT), zinc transporter, and cytokine genes to a modest (15 mg of Zn per day) dietary zinc supplement in human subjects. Transcript abundance was measured by quantitative real-time RT-PCR (QRT-PCR). Zinc supplementation increased MT mRNA abundance by up to 2-fold in RNA from leukocyte subsets, and 4-fold in RNA from DBS. Transcript levels for the zinc transporter genes ZnT1 and Zip3 were increased and decreased, respectively, by zinc supplementation. Expression of the ZnT and Zip genes among leukocyte subsets differ by up to 270-fold. Monocytes and granulocytes from supplemented subjects were activated by LPS, whereas T lymphocytes were activated by mimicking antigen presentation. With zinc consumption, TNF-alpha and IL-1beta expression was greater in activated monocytes and granulocytes, and IFN-gamma mRNA levels were higher in activated T lymphocytes. These studies show that QRT-PCR is a tool to reliably measure transcript abundance for nutritionally responsive genes in human subjects, and that a small sample of whole dried blood, when appropriately collected, can be used as the source of total RNA for QRT-PCR analysis. The results obtained also show that zinc supplementation of human subjects programs specific leukocytic subsets to show enhanced cytokine expression upon activation by stimulators of immunity.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Comparison of MT mRNA levels in monocytes (M), T lymphocytes (T), and granulocytes (G) of control human subjects with levels in a human reference RNA (R). Values are means ± SD (n = 3).
Fig. 2.
Fig. 2.
Relative MT mRNA levels in leukocyte subsets isolated from human subjects supplemented with dietary zinc. Values are means ± SD (n = 3–6 per group). Statistical difference from control is indicated (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001). Control subjects (open circles) were given placebo for 16 days. Supplemented subjects (filled) were given Zn (15 mg/day) for 10 days (hatched bar) and placebo for 6 days (open bar). (A) Monocytes. (B) T lymphocytes. (C) Granulocytes.
Fig. 3.
Fig. 3.
Relative MT transcript levels in RNA from dried spots of whole blood (DBS) from human subjects supplemented with dietary zinc. Values are means ± SD (n = 3–6 per group). Statistically different from control (∗∗, P < 0.01; ∗∗∗, P < 0.001). Control subjects (open circles) were given placebo for 16 days. Supplemented subjects (filled circles) were given Zn (15 mg/day) for 10 days (hatched bar) and placebo for 6 days (open bar).
Fig. 4.
Fig. 4.
Relative ZnT and Zip transcript levels in RNA from dried spots of whole blood (DBS) from human subjects supplemented with dietary zinc or the placebo. (A) Pooled DBS from subjects that were supplemented for 8 days. (B and C) Control subjects (open circles) were given placebo for 16 days. Supplemented subjects (filled circles) were given Zn (15 mg/day) for 10 days (hatched bar) and placebo for 6 days (open bar). Values are means ± SD (n = 3–4 per group). Statistically different from control (∗, P < 0.05; ∗∗∗, P < 0.001). (B) ZnT1 mRNA. (C) Zip3 mRNA.
Fig. 5.
Fig. 5.
Relative selected cytokine, MT, and ZnT1 transcript levels in nonactivated and activated monocytes, T lymphocytes, and granulocytes from human subjects supplemented with dietary zinc. Values are means ± SD (n = 3–6 per group). Within each panel, statistical differences at P < 0.05 are indicated by a different superscript. In vitro activation was assessed for monocytes, T lymphocytes, and granulocytes by TNF-α (A), IFN-γ (B), and IL-1β (C) expression, respectively. MT transcripts for these leukocyte subsets (DF, respectively) are also shown. ZnT1 transcripts are in GI, respectively. The cells were obtained from human subjects who were given Zn (15 mg/day) or a placebo for 4 days. The cells were then activated in vitro.

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