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Title:   Author  item
                                            Andree, Karlitem
                                            KIM, JIHYE - CHORIitem
                                            Kirschke, Catherineitem
                                            GREGG, JEFF - UNIV OF CALIF DAVISitem
                                            PAIK, HEEYOUNG - CHORIitem
                                            JOUNG, HYOJEE - CHORIitem
                                            Woodhouse, Leslieitem
                                            KING, JANET - CHORIitem
                                            Huang, Liping
  Submitted to: Journal of Nutrition
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 4/30/2004
                                    Publication  Date: 6/30/2004
                                    Citation: Andree, K.B., Kim, J., Kirschke, C.P., Gregg, J.P., Paik, H., Joung, H., Woodhouse, L.R., King, J., Huang, L. 2004. Investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans. Journal of Nutrition.  Journal of Nutrition. 134:1716-1723, 2004.Interpretive Summary: Human metabolism requires zinc for many vital biochemical activities. Zinc is associated with immune functions, spermatogenesis, and is a required element for the function of many enzymes. Although zinc deficiency and toxicity occur under only extreme conditions it has been indicated that many human populations suffer from marginal deficiency of this important trace metal. While the signs of severe zinc deficiency are obvious, assessing marginal zinc deficiency is difficult due to lack of clinical signs and a reliable laboratory test. Because of the importance of maintaining proper zinc levels for normal metabolism a reliable assay for the assessment of marginal zinc deficiency has been sought by scientists and clinicians involved in trace metal metabolism research. Different approaches have been used to develop an assay to detect marginal zinc deficiency. Previous studies have indicated that zinc levels found in human blood serum are under tight control and therefore don't fluctuate with changes in zinc intake. Other means of evaluating the status of zinc sufficiency in humans, such as measuring enzyme activity, have been found to be non-specific or unreliable. Zinc absorption for humans occurs in the small intestine, though with decreasing efficiency as age increases. After zinc is absorbed into the blood it is bound to blood serum proteins and later accumulates in the liver for redistribution to other organs. Several cellular proteins have been identified in humans that are involved in the transport and redistribution of zinc through storage of zinc, or in moving it into or out of cells. These specialized zinc transporters maintain intracellular zinc concentrations in a narrow range required for proper health.  Using a specific type of cultured white blood cells (B-lymphocytes) we found that expression of two zinc transport genes (ZNT1 and ZIP1) fluctuated in response to varying zinc levels in the culture media. The gene expression patterns of zinc transporters in white blood cells have been studied using quantitative RT-PCR analysis. This method involves measuring how much a protein is used in cells indirectly by measuring how much the gene for a particular protein is expressed. The expression of one of the zinc transport genes, ZNT1, was observed to increase in white blood cells in response to increased zinc in the culture medium, while the expression of a second zinc transporter, ZIP1, decreased in response to excess zinc in the culture medium. Following this finding we have examined the expression of these two genes, identified in our culture assays, using blood samples collected from human subjects. Two groups of Korean women (fifteen at age 20-25 years and fifteen at age 64-75 years) were chosen to validate the potential biomarkers of zinc status found in the gene expression results with cultured white blood cells. The different age groups used in our study may also provide some affirmation of the reports of decreased efficiency of zinc absorption with increasing age, as mentioned above.
The reduction of the ZIP1 expression in cultured white blood cells in response to increased extracellular zinc was duplicated in the white blood cell samples collected from the human subjects before and after taking a dietary zinc supplement. However no correlation of change in ZNT1 expression with increased dietary zinc was seen with the human subjects. We believe the difference between the results from our cell culture and the human subjects is due to the diversity of white blood cells in whole human blood versus a single blood cell type such as we grew in culture. In a future study we will separate blood cells into their specific subpopulations to analyze gene expression from separate white blood cell types to identify any correlations with zinc status. This work should provide the means for development of a clinical assay for zinc s
                                Technical Abstract: A bioassay for zinc status in human has been sought due to the importance of zinc, an essential trace metal, for many divergent functions in the human body. A sensitive bioassay for zinc status in human is lacking. Microarray and quantitative RT-PCR analyses were used for gene expression profiling of 0- or 30  M ZnSO4-treated  lymphocytes with the purpose of developing a biomarker for zinc status in human. The gene expression patterns of zinc transporters in the  -lymphocytes have been studied using quantitative RT-PCR analysis. One of the zinc transport genes, ZNT1, was observed upregulated in  -lymphocytes in response to increased zinc in the culture medium, while a second zinc transporter, ZIP1, was downregulated in response to excess zinc in the culture medium. Two groups of Korean women (fifteen at age 20-25 years and fifteen at age 64-75 years) were chosen to validate the potential biomarkers of zinc status found in the gene expression profiling with cultured  lymphocytes. The reduction of the ZIP1 expression in cultured  lymphocytes to increased extracellular zinc was duplicated in the whole white blood cell samples collected from the human subjects before and after taking zinc supplement.


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