Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/24/2002
Publication Date: 9/24/2002
Citation: Etcheverry, P., Wallingford, J.C., Miller, D.D., Glahn, R.P. 2002. Simultaneous determination of 45calcium and 65zinc uptake by caco-2 cells. Journal of Agricultural and Food Chemistry. 50:6287-6294. Interpretive Summary: One of the ways of studying the bioavailability of minerals in foods is to "label" the samples with radioactive forms (i.e. radioisotopes) of the mineral of study. Radioactive forms of minerals give off energy that can be measured with instruments known as "counters." Studies with humans, animals and cell cultures often use radioactive minerals which can be safely used at low levels. With radioisotopes one can track the mineral as it is absorbed, distributed, stored, and excreted. Measuring two radioisotopes simultaneously, however, is not a simple procedure. In this study, we describe a method for simultaneously determining two radioisotopes, 45Ca and 65Zn, in cultured human intestinal epithelial cells, which could also be adapted to human or animal studies. The methodology described in this paper utilizes two specific types of counters, gamma and liquid scintillation counters, both of which are commonly used in research. This methodology is relatively simple and should enable more nutritionists and food scientists to simultaneously the assess the bioavailability of these two minerals, thus increasing the productivity of this area of research.
Technical Abstract: We describe a simple method for simultaneously determining cell associated Ca and Zn in Caco-2 cells. Calcium and zinc uptake was measured via radioisotopes 45Ca and 65Zn. Preliminary studies revealed that 65Zn, a positron (+) and gamma emitter, contributed to 45Ca counts in a liquid scintillation counter (LSC). However, 45Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gamma C). In order to differentiate the counts of 45Ca from those of 65Zn, first a 65Zn labeled cell suspension was read in gamma C and a LSC, thus obtaining the relationship between the radioactive counts obtained from the gamma C and LSC. This information defined the linear relationship between gamma C 65Zn CPM and LSC65Zn CPM. Since the 45Ca and 65Zn counts obtained in the LSC are additive, giving Total LSC CPM, the value of LSC 45Ca CPM was obtained by subtracting LSC 65Zn CPM from Total LSC CPM for the dual labeled cell sample, obtaining then LSC 45Ca CPM. To determine the absolute activity or DPM of each isotope in the dual labeled sample, the linear relationship between DPM and CPM was determined for each isotope. We consider this method to be a simple and straightforward method for the determination of 45Ca counts from a sample also containing 65Zn, using gamma and liquid scintillation counters.