COMPETITIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION SHOWS THAT DIETARY ZINC SUPPLEMENTATION IN HUMANS INCREASES MONOCYTE METALLOTHIONEIN MESSENGER-RNA LEVELS

Zinc status is difficult to evaluate in humans. Metallothionein gene e xpression is transcriptionally regulated by dietary zinc and thus coul d serve as an assessment parameter based on zinc-dependent function. W e used semiquantitative reverse transcriptase-polymerase chain reactio n (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 NycoP rep(TM) 1.068. Monocyte purity was determined by flow cytometry using fluorescent antihuman 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 p roportional to MT mRNA levels in monocytes from zinc-supplemented and control subjects. RT-PCR oligonucleotide primers were designed to ampl ify 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 cD NA quantitation. The RT-PCR data show that there was a significant inc rease in monocyte MT mRNA in subjects within 6 d of zinc supplementati on, which remained elevated at d 15 of supplementation. In contrast, p lasma zinc was greater at d 6 of zinc supplementation, but by d 15 of supplementation, while still elevated, was close to control levels. Th ese data suggest that monocyte MT mRNA levels respond to zinc suppleme ntation and that the response could serve as a more useful assessment variable than plasma zinc for the measurement of zinc status in humans .