Zinc flexes its muscle: Correcting a novel analysis of calcium for zinc interference uncovers a method to measure zinc

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.

Figure 1.

Total concentration of zinc or calcium was determined in acid digests of rat soleus or plantaris muscle tissue by ICP-MS. Results are expressed in mmole/kg (muscle wet weight). *, significantly different when Zn²⁺ is compared with Ca²⁺ in the soleus muscle; P < 0.05; Student’s unpaired t test. ***, significantly different when Zn²⁺ is compared with Ca²⁺ in the plantaris muscle, and when Zn²⁺ in the soleus is compared with Zn²⁺ in the plantaris; P < 0.0001; Student’s unpaired t test. No significant difference was found when comparing Ca²⁺ in the soleus with Ca²⁺ in the plantaris muscle. n = 5 for each muscle type. Error bars represent mean ± SEM.

Figure 2.

TPEN and BAPTA spectrophotometric analysis. BAPTA absorbance shift after Zn²⁺ or Ca²⁺ binding in the presence or absence of TPEN was determined by spectrophotometric analysis (250–350-nm scan). (A) The spectrum of 0.2 mM TPEN in the measurement solution shown as the blue solid line. The spectrum of 0.15 mM BAPTA in the measurement solution shown as the black solid line. The combination of 0.15 mM BAPTA and 0.2 mM TPEN spectrum shown as the black dash/dot line. (B) The addition of 0.15 mM ZnCl₂ into 0.15 mM BAPTA in the measurement solution caused a reduction in BAPTA absorbance (blue solid line) compared with the unbound BAPTA spectrum around 292 nm (black solid line). The addition of 0.2 mM TPEN into the Zn²⁺-bound BAPTA yielded a spectrum shown as the black dash/dot line. (C) Determination of the extinction coefficient (Δε) for Ca²⁺ or Zn²⁺ binding to BAPTA: Ca²⁺ standard or ZnCl₂ was added step-wise to the 0.15-mM BAPTA solution to obtain 0.01-, 0.025-, 0.05-, 0.075-, 0.1-, 0.15-, and 0.2-mM final concentrations. The Ca²⁺ and Zn²⁺ additions to BAPTA were repeated twice. Determination of Δε for Ca²⁺ or Zn²⁺ binding to BAPTA in the presence of TPEN: TPEN stock solution was added into 0.15 mM BAPTA in the measurement solution to reach a final concentration of 0.2 mM. Ca²⁺ standard or ZnCl₂ was added step-wise to the 0.15-mM BAPTA/0.2-mM TPEN solution to obtain 0.01-, 0.025-, 0.05-, 0.075-, 0.1-, 0.15-, and 0.2-mM final concentrations. The Ca²⁺ and Zn²⁺ additions to 0.15 mM BAPTA/0.2 mM TPEN were repeated twice. Values of BAPTA absorbance at 292 nm were plotted using GraphPad Prism software as a function of increasing Ca²⁺ or Zn²⁺ concentration. The linear portion of the additions (0–0.10 mM) was used to estimate the extinction coefficients by performing linear regression analysis.

Figure 3.

Representative absorbance scans obtained for total calcium and zinc determinations using the BAPTA method. See Results for additional explanation. This particular example is from rat soleus muscle tissue homogenate and yielded the following values for total calcium and zinc concentrations (2.738 mmole/kg Ca²⁺ without TPEN addition; 2.348 mmole/kg Ca²⁺ with TPEN addition). (A) Blank measurement solution. (B) Rat soleus muscle tissue homogenate. 0.15 mM BAPTA, 1 mM EGTA, 1 mM CaCl₂, and 0.2 mM TPEN were used in the corresponding trials. A vertical dotted line is used to help visualize the absorbance values at 292 nm.