title: Competitive reverse transcriptase-polymerase chain reaction shows that dietary zinc supplementation in humans increases monocyte metallothionein mRNA levels.
abstract: Zinc status is difficult to evaluate in humans. Metallothionein gene expression is transcriptionally regulated by dietary zinc and thus could serve as an assessment parameter based on zinc-dependent function. We used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to establish that MT mRNA is increased in a human monocytic cell line by addition of zinc to the medium. To examine this response in human subjects, a dietary supplement of 50 mg zinc gluconate/d was given for 15 d. Monocytes were purified from venous blood using NycoPrep 1.068. Monocyte purity was determined by flow cytometry using fluorescent anti-human monocyte CD14 antibodies. Total monocyte RNA was extracted and converted to cDNA by reverse transcription. Competitive RT-PCR was used to analyze differences between cDNA levels that are proportional to MT mRNA levels in monocytes from zinc-supplemented and control subjects. RT-PCR oligonucleotide primers were designed to amplify both a 201 bp segment of the human MT cDNA and a 180 bp competitor cDNA template. The 180 bp competitor cDNA template was used for MT cDNA quantitation. The RT-PCR data show that there was a significant increase in monocyte MT mRNA in subjects within 6 d of zinc supplementation, which remained elevated at d 15 of supplementation. In contrast, plasma zinc was greater at d 6 of zinc supplementation, but by d 15 of supplementation, while still elevated, was close to control levels. These data suggest that monocyte MT mRNA levels respond to zinc supplementation and that the response could serve as a more useful assessment variable than plasma zinc for the measurement of zinc status in humans.
authors.affiliation: Food Science and Human Nutrition Department, Center for Nutritional Sciences, University of Florida, Gainesville 32611, USA.
authors.forename: V K
authors.initials: VK
authors.lastname: Sullivan
authors.forename: R J
authors.initials: RJ
authors.lastname: Cousins
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D001483:Base Sequence; D015152:Blotting, Northern; D002469:Cell Separation; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry / Q000379:methods; D005527:Food, Fortified; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007951:Leukemia, Myeloid / Q000097:blood / Q000378:metabolism / Q000473:pathology; D008297:Male; D008668:Metallothionein / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D009000:Monocytes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000008:administration & dosage / Q000097:blood / Q000494:pharmacology*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D008668:Metallothionein; D012194:RNA-Directed DNA Polymerase; D015032:Zinc