Early research into human copper metabolism involved studies of copper intake and excretion, copper balance and tissue concentrations, which permitted the estimation of bodily requirements and dietary recommendations.104 Studies on laboratory animals, and the use of in vitro techniques such as intestinal perfusion and the creation of sacs from everted duodenal segments, have contributed much to our understanding of intestinal mechanisms. The use of balance studies to examine copper metabolism poses certain difficulties. Firstly, the regulation of copper absorption according to dietary intake is a process which may require a period of adaptation. For absorption to reflect bodily requirement accurately, therefore, a balance study must be of considerable duration.92 Secondly, the estimation of losses is difficult owing to a current scarcity of data concerning copper levels in sweat, integument, hair, nails, menstrual blood and semen. Thirdly, balance studies of children must account for the changing copper requirements associated with growth, about which little information is available.105
The introduction of isotopic tracers as an investigative tool has permitted detailed examination of absorption mechanisms, dose effects and interactions with other minerals and food components. Findings in mammals of a saturable, carrier-mediated transport mechanism are compatible with the dose-related reduction in absorption which has been demonstrated in humans. The absorption of stable and radioactive isotopes of copper may be determined after oral administration by monitoring either their disappearance from the gut lumen or their incorporation into biomolecules.106 Copper has seven radioisotopes, of which only 64Cu and 67Cu have half-lives long enough to be useful in metabolic research - 12.8 h and 58.5h respectively. These relatively short half-lives limit the use of radioisotopes to short-term studies.
Longer-term studies require the use of stable isotopes which have several additional advantages: because they emit no radiation, they are safe to use in high-risk population groups; and because there is no decay, samples can be stored without loss of signal. Copper has two stable isotopes, 63Cu and 65Cu, which both have high natural abundances - 69.2% and 30.8% respectively. To act as a tracer, an isotope must be 'enriched' to a higher proportion than in nature. The production of enough 63Cu or 65Cu to detect above background levels is costly. The use of such large doses raises further problems. The intravenous administration of non-physiological quantities of the mineral may alter normal metabolism107 while the labelling of food with 65Cu has been found to change its copper content substantially.108 In studies of trace minerals, the simultaneous use of multiple stable isotopes offers a means to study the effects of different compounds and different routes of administration. Such studies are necessarily impossible for copper, because with only two stable isotopes, only one can be enriched at a time.
Biological samples obtained in stable isotope studies are analysed by determining isotopic ratios. Available methods are generally slow and expensive, and require access to sophisticated analytical equipment. Neutron Activation
Analysis, Electron Ionisation Mass-Spectrometry and Gas-Chromatography Mass-Spectrometry all offer relatively poor precision, while Thermal Ionisation Mass-Spectrometry is laborious and slow. Inductively Coupled Plasma Mass-Spectrometry, used since the 1980s for trace-element quantification, offers acceptable precision with faster analysis and a lower limit of detection than the other methods.109 Most radioisotopes can be measured by whole-body counting of gamma-emissions, but this method of detection is not readily applicable to copper, owing to the radioisotopes' short half-life. It has, however, been applied in studies of abnormal copper absorption and retention.110
Faecal monitoring, of stable or radioisotopes, is currently the most widely used method for assessing copper absorption. A stool marker may be given simultaneously to test for completeness of faecal collection, and may consist of indigestible beads or a non-absorbable chemical marker. In this method, the relatively rapid re-excretion of absorbed copper necessitates special consideration. Even before the non-absorbed fraction of an oral dose has left the body - a process which has been found to take five to seven days - re-excretion of the absorbed isotope will have begun. To correct for this, the rate of endogenous excretion must be determined. Owing to the large inter-individual variation,110 it should be measured in each individual. Faecal monitoring of a radioisotope for longer than five days would require more than the maximum safe dose106 so endogenous excretion must be determined on a separate occasion using an intravenous dose.
One application of tracer data obtained from isotope studies is the development of compartmental models of metabolism. In this technique, modelling software is used to compile extensive data on copper distribution and transport into a model simulating whole-body copper metabolism. This provides a powerful tool to describe and predict copper kinetics and to determine dietary require-ments.111 Kinetic modelling provides a means to correlate experimental data from previous studies. Existing information including tissue concentrations, fractional transfer and turnover rates can be assembled into a system in which known components are viewed in perspective. This can have the effect of highlighting areas requiring further research. It can also be used to improve experimental design by simulating in advance the system of interest.
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