Zinc Status Assessment by Analysis of Mononuclear Cell Metallothionein mRNA Using Competitive-Reverse Transcriptase-Polymerase Chain Reaction

  • Lee, Soo-Lim (Department of Food Science and Nutrition, Andong National University) ;
  • Yoon, Jin-Sook (Department of Food Science and Nutrition, Keimyung University) ;
  • Kwon, Chong-Suk (Department of Food Science and Nutrition, Andong National University) ;
  • Beattie, John H. (Cellular Integrity Division, Rowett Research Institute, Aberdeen, Scotland, United Kingdom) ;
  • Kwun, In-Sook (Department of Food Science and Nutrition, Andong National University)
  • Published : 2004.09.01


Marginal Zn deficiency is prevalent through the world and yet human zinc status has not been properly assessed due to the lack of a reliable diagnostic indicator. One potential possibility for zinc status assessment using Zn-binding protein, metallothionein (MT)-mRNA, has been proposed. The purpose of the present study was aimed to show whether measurement of mononuclear cell (MNC) MT mRNA, using a competitive-reverse transcriptase-polymerase chain reaction (competitive-RT-PCR) assay, could indicate zinc status in human subjects. In this study, MNC MT-mRNA expression was measured using a competitive-RT-PCR to compare before and after 14 days of zinc supplementation (50 mg Zn/das zinc gluconate). RT-PCR oligonucleotide primers which were designed to amplify both a 278 bp segment of the human MT-2A cDNA and a 198 bp mutant competitor cDNA template from MNCs, were prepared. MT-2A mRNA was normalized by reference to the housekeeping gene, $\beta$-actin, mRNA for which was also measured by competitive-RT-PCR. There was considerable inter-individual variation in MT-mRNA concentration and yet, the mean MT-2A mRNA level increased 4.7-fold after Zn supplementation, as compared to before Zn supplementation. This MT-2A mRNA level was shown as the same pattern and, even more sensitive assay, compared to the conventional plasma and red blood cells (RBCs) Zn assessment in which plasma and RBCs zinc levels increased 2.3- and 1.2-fold, respectively (p<0.05). We suggest that MT competitive-RT-PCR can be a useful assessment tool for evaluating human zinc status.


  1. Hambidge KM, Casey CE, Crebs NF. 1986. Zinc. In Trace elements in human and animal nutrition. 5th ed. Mertz W, ed. Academic Press, Orlando, FL, USA. Vol 2, p 1-137
  2. Hambidge KM, King JC, Kern DL, English-Wstcott JL, Stall C. 1990. Prebreakfast plasma Zn concentrations: The effect of previous meals. J Trace Elem 4: 229-231
  3. Chester JK. 1997. Zinc. In Handbook of nutritionally essential mineral elements. O'Dell BL, Sunde RA, eds. Marcel Dekker, Inc., New York
  4. King JC. 1990. Assessment of zinc status. J Nutr 120: 1474-1479
  5. Allan AK, Hawksworth GM, Woodhouse LR, Sutherland B, King JC, Beattie JH. 2000. Lymphocyte metallothionein mRNA responds to marginal zinc intake in human volunteers. Brit J Nutr 84: 747-756
  6. Sullivan VK, Cousins RJ. 1997. Competitive reverse-transcriptase-polymerase chain reaction shows that dietary zinc supplementation in humans increases mononuclear cell metallothionein mRNA levels. J Nutr 127: 694-698
  7. Sullivan VK, Burnett FR & Cousins RJ. 1998. Metallothionein expression is increased in mononuclear cells and erythrocytes of young men during zinc supplementation. J Nutr 128: 707-713
  8. Grider A, Bailey LB & Cousins RJ. 1990. Erythrocyte metallothionein as an index of zinc status in humans. Proc Natl Acad Sci USA 87: 1259-1262
  9. Thomas EA, Bailey IB, Kauwell GA, Lee DY, Cousins RJ. 1992. Erythrocyte metallothionein response to dietary zinc in humans. J Nutr 122: 2408-2414
  10. Harley DB, Menon CR, Rachubinski RA, Nieboer E. 1989. Metallothionein mRNA and protein induction by cadmium in peripheral-blood leukocytes. Biochem J 262: 837-879
  11. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium. Anal Biochem 162: 156-159
  12. Gilliland G, Perrin S, Blanchard K, Bunn HF. 1990. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 87: 2725-2729
  13. Beattie JH, Kwun IS. 2004. Is zinc deficiency a risk factor or atherosclerosis? Brit J Nutr 91: 177-181
  14. Cousins RJ. 1996. Zinc. In Present knowledge in nutrition. 7th ed. Filer LJ, Ziegler EE, eds. International Life Sciences Institute Nutrition Foundation, Washington, DC. p 293-306
  15. Trayhurn P, Duncan JS, Wood AM, Beattie JH. 2000 (a). Metallothionein gene expression and secretion in white adipose tissue. Am J Physiol Regul Integr Comp Physiol 279: R2329-2335
  16. Trayhurn P, Duncan JS, Wood AM, Beattie JH. 2000 (b). Regulation ofmetallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture. Horm Metab Res 32: 542-547
  17. Beattie JH, Wood AM, Trayhurn P, Jasani B, Vincent A, McCormack G, West AK. 2000. Metallothionein is expressed in adipocytes of brown fat and is induced by catecholamines and zinc. Am J Physiol Regul Integr Comp Physiol 278: R1082-1089
  18. Sato M, Mehra RK, Bremner I. 1984. Measurement of plasma metallothionein-1 in the assessment of the zinc status of zinc-deficient and stressed rats. J Nutr 114: 1683- 1689
  19. Bremner I. 1987. Nutritional and physiological significance of metallothionein. In Metallothionein II. Kagi JHR, Kojima Y, eds. Birkhauser Verlag, Basel/Boston. p 81-87
  20. Cousins RJ. 1994. Metal elements and gene expression. In Annual review of nutrition. Olson RE, ed. Annual Reviews, Palo Alto, CA, USA. p 449-469
  21. Dunn MA, Blalock TL, Cousins RJ. 1987. Metallothionein. Proc Soc Exp Biol Med 185: 107-119
  22. Hamer DH. 1986. Metallothionein. Ann Biochem 55: 913- 951
  23. Blalock TL, Dunn MA, Cousins RJ. 1988. Metallothionein gene expression in rats: tissue-specific regulation by dietary copper and zinc. J Nutr 118: 222-228
  24. Cousins RJ, Lee-Ambrose LM. 1992. Nuclear zinc uptake and interactions and metallothionein gene expression are influenced by dietary zinc in rats. J Nutr 122: 56-64