Abstract
The divalent cation chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), often used to buffer physiological changes in cytosolic Ca(2+), also binds Zn(2+) with high affinity. In a recently published method (Lamboley et al. 2015. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201411250), the absorbance shift of BAPTA at 292 nm was successfully used to determine the total calcium concentrations of various skeletal muscle tissues. In the present study, we show that endogenous Zn(2+) in rat skeletal muscle tissue can be unknowingly measured as "Ca(2+)," unless appropriate measures are taken to eliminate Zn(2+) interference. We analyzed two rat skeletal muscle tissues, soleus and plantaris, for total calcium and zinc using either inductively coupled plasma mass spectrometry (ICP-MS) or the BAPTA method described above. ICP-MS analysis showed that total zinc contents in soleus and plantaris were large enough to affect the determination of total calcium by the BAPTA method (calcium = 1.72 ± 0.31 and 1.96 ± 0.14, and zinc = 0.528 ± 0.04 and 0.192 ± 0.01; mean ± standard error of the mean [SEM]; n = 5; mmole/kg, respectively). We next analyzed total calcium using BAPTA but included the Zn(2+)-specific chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) that buffers Zn(2+) without affecting Ca(2+)/BAPTA binding. We found that estimated concentrations of total calcium ([CaT]WM) in soleus and plantaris were reduced after TPEN addition ([CaT]WM = 3.71 ± 0.62 and 3.57 ± 0.64 without TPEN and 3.39 ± 0.64 and 3.42 ± 0.62 with TPEN; mean ± SEM; n = 3; mmole/kg, respectively). Thus, we show that a straightforward correction can be applied to the BAPTA method to improve the accuracy of the determination of total calcium that should be applicable to most any tissue studied. In addition, we show that using TPEN in combination with the BAPTA method allows one to make reasonable estimates of total zinc concentration that are in agreement with the direct determination of zinc concentration by ICP-MS.