In the next study, we assessed the effect of the Upper Level (UL) of dietary recommendations in apparently healthy adults (4, 5, 8). The UL value is a concept developed for regulatory purposes to define safe limits of intake for the normal human. It is the level of intake from food, water and supplements that is unlikely to pose risks of adverse health effects from excess over the long term, in apparently healthy individuals, in an age- and sex-specific population group (5). Although Cp is not a good indicator when infectious or inflammatory processes are present, it is one of the indicators used clinically to detect alterations of copper metabolism _ for example, in patients suspected to suffer Wilson's disease (12). We decided to explore the capacity of this protein to show differential responses to copper intake among normal individuals. We hypothesized that healthy individuals would respond to Cu in accordance with their place in the serum Cp distribution curve, considering a lower value as an index of long-term lower intake (4). Thus, two study groups were formed considering the individual serum Cp values (low Cp and high Cp, n= 41 per group). The study was a prospective controlled trial; participants (women and men, 18-50 years) belonged to the upper and lower decile of the Cp distribution curve obtained in 800 healthy adults. Participants ingested a single daily dose of 10 mg Cu/d for 60 days. Before and immediately after the Cu supplementation period, blood (serum Cu, Cp protein, homocysteine, liver GOT, GPT and GGT, Zn-Cu-SOD activity in erythrocytes, glutathione and IL-2 production and SOD and CCS mRNA expression in peripheral mononuclear cells, and HCTR1 and DMT1 mRNA expression in monocytes) and urine (urinary Cu excretion after DMPS _ Dimaval^R _ challenge) studies were performed.

In an additional analysis aimed at identifying factors that may modulate the response to copper exposure, Principal Component Analysis and Linear Discriminant Analysis were used to assess blood and urinary Cu indicators that showed a differential response to Cu, as a function of sex (8). This analysis revealed that individual Cp values in serum reliably differentiate subgroups within the normal population in their response to excess Cu. The responses detected were dependant both on the Cp values and on sex: women with higher and men with lower Cp values exhibited the greatest response to excess Cu.

In a subsequent study, we further explored the effect of chronic excess copper exposure in apparently healthy adult men who received 8 mg Cu/day for 6 months. Preliminary results showed no detectable changes in liver enzymatic activities nor in indicators of oxidative stress, indicating that administration of 8 mg Cu/day for 6 months is handled adequately by homeostatic mechanisms. ANOVA analysis for repeated measures confirmed that the variable copper (dosing) was not related significantly to the indicators measured; however, there was a significant association between the variable time and the indicators measured, suggesting that seasonality is a factor that influences the responses to copper exposure.

To learn about the impact of an acute dose of copper on the indictors that evaluate copper status, liver function and oxidative stress were explored during the same 6-month protocol. A single, 10 mg Cu (as copper sulfate) or placebo dose was administered to a subgroup of participants immediately before and after finishing the protocol. After 12 hrs, 1, 3 and 7 days of dosing, blood samples were drawn and indicators were measured. Preliminary results show that curves of serum LDH activity in the experimental individuals were no different in comparison to those of the placebo subjects during the 7-day evaluation.

In summary, these studies shed light on the effects of chronic and acute copper dosing in healthy adults. In the chronic studies, the administration of 10 mg Cu/d mixed with food for two months did not induce adaptive responses, but 10 mg Cu/d administered as a single dose per day (mimicking a daily bolus) during two months provoked a transient response of liver enzyme activities and of peripheral mononuclear cell glutathione. A dose of 8 mg Cu per day for 6 months did not trigger detectable responses but showed that seasonality is likely to be another variable that influences the responses observed. Considering that 10 mg of copper per day represents an Upper Limit for human chronic consumption of copper, further studies should clarify the safety of this figure when different regimens are used. In the acute studies, the differences observed