This is the first study to demonstrate that CCS protein expression responds to mild Cu deficiency induced by moderately high dietary Zn, underscoring the potential usefulness of CCS as a biomarker in a clinical setting. In contrast to rats and mice used in previous studies [19, 21, 22], rats used in this study had normal haematology and did not show any indications of the development of anaemia, indicating that these rats were not overtly Cu deficient. Further, rats did not show impaired growth, a phenotype associated with severe Cu deficiency in growing rats .
Erythrocyte CCS expression was determined to be the most sensitive index of reduced Cu nutriture and was able to detect a decrease in Cu status in rats fed only twice the AIN recommended amount of Zn . This sensitivity may also partly account for the absence of a significant difference in erythrocyte CCS expression between responders and non-responders for plasma Cu, as CCS may have increased in some rats fed high Zn but characterise as non-responders. Notably, erythrocyte CCS expression in non-responders was higher than in Zn-30 rats and this difference was approaching statistical significance (P = 0.09). Liver CCS also responded to elevated dietary Zn and was increased in rats fed 4 times the recommended amount. These are important findings, as these same rats did not show a significant decrease in SOD1 activity, the biomarker of reduced Cu status used to set the ULs for Zn.
Rats fed elevated Zn showed an all or none response for decreased plasma Cu and Cp activity, indicating that these parameters are not ideal for diagnosing small reductions in Cu status and providing an accurate assessment of precise Cu nutriture. These data are consistent with a previous study showing a similar all or none response for Cp activity in rats fed elevated levels of Zn . This response likely reflects the presence of strong homeostatic mechanisms that maintain plasma Cu levels in the normal range until liver stores have been appreciably depleted.
Although a gold standard for assessing reductions in Cu status is lacking, decreased liver Cu content is regarded as one of the most accurate and sensitive measures in experimental animals, while plasma Cu and Cp activity are the most common indicators used in clinical situations. The strong inverse correlation of liver CCS expression with these markers indicates that increased CCS expression was specific for reduced Cu status and is a good predictor of changes in common measures of Cu deficiency. Erythrocyte CCS showed a weaker association with these indicators that may be explained, in part, by the slow turnover of erythrocytes, which may not accurately reflect the current Cu status of the rats. This is in contrast to plasma and liver Cu levels and Cp activity that likely respond more rapidly to changes in Cu availability.
During our experiments we discovered that CCS protein in WBCs is more susceptible to degradation than CCS in erythrocytes, requiring that WBCs be isolated quickly from blood samples. Samples showing degradation of CCS were excluded from our analyses. Of the 7 rats per diet group analysed, we noticed that the majority of rats fed high Zn were characterised as non-responders for decreased plasma Cu and Cp activity. This over sampling of rats showing normal plasma Cu and Cp activity may account for the absence of a significant increase in WBC CCS in rats fed elevated Zn. Nonetheless, WBC CCS expression was increased in rats fed the Cu-deficient diet and was highly correlated with conventional measures of Cu status. Further, rats characterised as responders for plasma Cu (which represents rats with poorer Cu status) had higher WBC CCS levels than non-responders or Zn-30 rats, indicating that WBC CCS responds to Cu deficiency induced by excess zinc. Because of the slow turnover of erythrocytes, WBC CCS may have value as an indicator of early reductions in Cu status. Interestingly, we found that the basal expression level of CCS in WBCs is much higher than in other tissues such as liver, heart and erythrocytes in rats (data not shown).
A major finding of this paper is that rats fed 8 times the recommended amount of Zn (Zn-240) showed overall better Cu status than rats fed 4 times the recommended amount (Zn-120). This was consistently observed with several biomarkers. Remarkably, Cu status indicators of most rats fed the Zn-240 diet were indistinguishable from rats fed a diet normal in Zn. Importantly, liver CCS was significantly lower in Zn-240 compared to Zn-120 rats, further demonstrating the exquisite sensitivity of CCS to changes in Cu status. At this point, we cannot offer a definite explanation as to why other studies from our laboratory [9, 34] failed to detect this biphasic response, other than to speculate that differences in the bioavailability of the Zn source, other dietary components or duration of the studies may account for this discrepancy.
Presently, the mechanism by which Zn interferes with Cu absorption is unknown, although it is believed to occur at the site of the intestinal enterocyte. It was thought that increased metallothionein (MT) levels sequestered Cu in the enterocyte leading to Cu deficiency [35, 36]. However, MT-null mice fed elevated Zn become Cu deficient indicating that MT induction is not the primary cause of the Cu deficiency . Results presented here are consistent with these data, as rats fed the Zn-60 and Zn-120 diet showed reduced Cu status in the absence of increased duodenal MT-1 transcript. Because MT can bind Cu, however, it would be interesting to determine whether higher MT-1 expression in Zn-240 rats played any role in increasing Cu in the intestinal mucosa and improving Cu status of these rats compared to Zn-120 rats.
Experiments with Caco-2 cells have indicated that elevated levels of Zn affect Cu transport and reduce Cu efflux at the basolateral side , suggesting that Zn may block absorption of Cu by affecting the activity of a Cu transporter. Thus, improved Cu status of Zn-240 compared to Zn-120 rats may reflect a secondary effect of the higher Zn diet on the activity of a Cu transporter. It is known that distinct pools of Zn exist within cells and changes in expression of Zn-trafficking proteins can alter the intracellular distribution of Zn . For example, increased expression of ZnT-2 likely promotes the accumulation of Zn within vesicles, decreasing cytoplasmic Zn levels . In addition, increased MT levels result in more Zn bound to MT. Given that transcripts of several Zn-trafficking proteins were increased in the duodenum of rats fed the Zn-240 but not the Zn-120 diet, it is possible that increased expression of one or more of these Zn-trafficking proteins altered the distribution of Zn within absorptive enterocytes of Zn-240 rats in a manner that alleviated the block in activity of a Cu transporter.
Lastly, we report an interesting observation that rats fed the highest level of Zn had increased body weight. Although it is well recognized that Zn deficiency can result in impaired growth , it is not established that consuming larger quantities of Zn, above recommend amounts, can enhance growth. Given that lower doses of Zn above the AIN recommended amount clearly depressed Cu status of rats, the benefit of consuming this higher level of Zn is presently unclear.