Assessment of zinc status by assay of human metallothionein mRNA in T-lymphocytes
Diagnosis of marginal zinc deficiency using current biochemical markers is unreliable due the lack of sensitivity or specificity. Metallothionein (MT) levels in various fluids and tissues have been shown to reflect zinc status, but measurement of the protein has proved difficult in plasma and white blood cells. An alternative to measuring MT protein is the assay of MT mRNA in blood cells. Northern blotting was not sensitive enough to detect basal levels of MT from human T cells. A sensitive RTPCR assay was developed which detected and measured human MT-2A mRNA in T cells. A competitive MT-2A DNA standard with an 80 base-pair deletion was constructed that co-amplified with the MT-2A mRNA during RT-PCR. After analysis of the PCR products by gel electrophoresis, the ratio of the added standard to MT-2A was used to quantify the MT-2A mRNA expression. Competitive RT-PCR was also carried out for β-actin to correct for RNA degradation and loading. Capillary electrophoresis combined with laser-induced fluorescence detection can also allow sensitive and rapid analysis of RT-PCR reactions. The RT-PCR assay was used to measure MT-2A gene expression in T cells from human volunteers in zinc deficiency studies. In a severe zinc deprivation study (>0.5 mg Zn per day) most of the samples were too degraded for accurate analysis, showing the requirement of a degradation control for quantitative RT-PCR. In a marginal zinc deprivation study, human volunteers were given a diet that was changed from 15mg Zn/day to 5mg Zn/day. After 50 days on the low zinc diet, each individual showed a decrease in T lymphocyte MT-2A mRNA levels (30-80% of baseline value). The T lymphocyte MT-2A mRNA levels increase upon zinc repletion (15mg Zn/day) after about two weeks. There were no apparent changes in plasma zinc concentration during the study. This suggests that T-lymphocyte mRNA measurements may be a sensitive index of zinc status in humans.