Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Shellaina  J.V. Gordon; Yao Xiao; Amanda  L. Paskavitz; Napole&#xF3;n Navarro-Tito; Juan  G. Navea; Teresita Padilla-Benavides

doi:10.3791/59519

JoVE Journal > Biochemistry

生物化学

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Published: May 16, 2019

doi:

10.3791/59519

Shellaina J.V. Gordon*¹, Yao Xiao*², Amanda L. Paskavitz³, Napoleón Navarro-Tito⁴, Juan G. Navea², Teresita Padilla-Benavides¹

¹Department of Biochemistry and Molecular Pharmacology,University of Massachusetts Medical School, ²Department of Chemistry,Skidmore College, ³Candiac MR Center,Beth Israel Deaconess Medical Center, ⁴Facultad de Ciencias Químico Biológicas,Universidad Autónoma de Guerrero

Summary

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Abstract

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson’s and Menkes’ diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Introduction

Transition and heavy metals, such as Zn, Cu, Mn, and Fe, are found naturally in the environment in both nutrients in food and pollutants. All living organisms require different amounts of these micronutrients; however, exposure to high levels is deleterious to organisms. Metal acquisition is mainly through the diet, but metals can also be inhaled or absorbed through the skin¹^,²^,³^,⁴^,⁵. It is important to note that the presence of metals in atmospheric particles is increasing and has been largely associated with health risks. Due to anthropogenic activities, increased levels of heavy metals such as Ag, As, Cd, Cr, Hg, Ni, Fe, and Pb have been detected in atmospheric particulate matter, rainwater, and soil⁶^,⁷. These metals have the potential to compete with essential physiological trace elements, particularly Zn and Fe, and they induce toxic effects by inactivating fundamental enzymes for biological processes.

The trace element Zn is redox neutral and behaves as a Lewis acid in biological reactions, which makes it a fundamental cofactor necessary for protein folding and catalytic activity in over 10% of mammalian proteins⁸^,⁹^,¹⁰; consequently, it is essential for diverse physiological functions⁸^,¹¹. However, like many trace elements, there is a delicate balance between these metals facilitating normal physiological function and causing toxicity. In mammals, Zn deficiencies lead to anemia, growth retardation, hypogonadism, skin abnormalities, diarrhea, alopecia, taste disorders, chronic inflammation, and impaired immune and neurological functions¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸. In excess, Zn is cytotoxic and impairs absorption of other essential metals such as copper¹⁹^,²⁰^,²¹.

Additionally, some metals like Cu and Fe have the potential to participate in harmful reactions. Production of reactive oxygen species (ROS) via Fenton chemistry can interfere with the assembly of iron sulfur cluster proteins and alter lipid metabolism²²^,²³^,²⁴. To prevent this damage, cells utilize metal-binding chaperones and transporters to prevent toxic effects. Undoubtedly, metal homeostasis must be tightly controlled to ensure that specific cell types maintain proper levels of metals. For this reason, there is a significant need to advance techniques for accurate measurement of trace metals in biological samples. In developing and mature organisms there exists a differential biological need for trace elements at the cellular level, at different developmental stages, and in normal and pathological conditions. Therefore, precise determination of tissue and systemic metal levels is necessary to understand organismal metal homeostasis.

Graphite furnace atomic absorption spectrometry (GF-AAS) is a highly sensitive technique used for small sample volumes, making it ideal to measure transition and heavy metals present in biological and environmental samples²⁵^,²⁶^,²⁷^,²⁸. Moreover, due to high sensitivity of the technique, it has been shown to be appropriate for studying the fine transport properties of Na⁺/K⁺-ATPase and gastric H⁺/K⁺-ATPase using Xenopus oocytes as a model system²⁹. In GF-AAS, the atomized elements within a sample absorb a wavelength of radiation emitted by a light source containing the metal of interest, with the absorbed radiation proportional to the concentration of the element. Elemental electronic excitation takes place upon absorption of ultraviolet or visible radiation in a quantized process unique for each chemical element. In a single electron process, the absorption of a photon involves an electron moving from a lower energy level to higher level within the atom and GF-AAS determines the amount of photons absorbed by the sample, which is proportional to the number of radiation absorbing elements atomized in the graphite tube.

The selectivity of this technique relies on the electronic structure of the atoms, in which each element has a specific absorption/emission spectral line. In the case of Zn, the absorbance wavelength is 213.9 nm and can be precisely distinguished from other metals. Overall, GF-AAS can be used to quantify Zn with adequate limits of detection (LOD) and high sensitivity and selectivity²⁵. The changes in absorbed wavelength are integrated and presented as peaks of energy absorption at specific and isolated wavelengths. The concentration of the Zn in a given sample is usually calculated from a standard curve of known concentrations according to the Beer-Lambert law, in which the absorbance is directly proportional to the concentration of Zn in the sample. However, applying the Beer-Lambert equation to GF-AAS analyses also presents some complications. For instance, variations in the atomization and/or non-homogenous concentrations of the samples can affect the metal measurements.

The metal atomization required for GF AAS trace elemental analysis consists of three fundamental steps. The first step is desolvation, where the liquid solvent is evaporated; leaving dry compounds after the furnace reaches a temperature of around 100 °C. Then, the compounds are vaporized by heating them from 800 to 1,400 °C (depending on the element to be analyzed) and become a gas. Finally, the compounds in the gaseous state are atomized with temperatures that range from 1,500 to 2,500 °C. As discussed above, increasing concentrations of a metal of interest will render proportional increases on the absorption detected by the GF-AAS, yet the furnace reduces the dynamic range of analysis, which is the working range of concentrations that can be determined by the instrument. Thus, the technique requires low concentrations and a careful determination of the dynamic range of the method by determining the LOD and limit of linearity (LOL) of the Beer-Lambert law. The LOD is the minimum quantity required for a substance to be detected, defined as three times the standard deviation of Zn in the matrix. The LOL is the maximum concentration that can be detected using Beer-Lambert law.

In this work, we describe a standard method to analyze the levels of Zn in whole cell extracts, cytoplasmic and nuclear fractions, and in proliferating and differentiating cultured cells (Figure 1). We adapted the rapid isolation of nuclei protocol to different cellular systems to prevent metal loss during sample preparation. The cellular models used were primary myoblasts derived from mouse satellite cells, murine neuroblastoma cells (N2A or Neuro2A), murine 3T3 L1 adipocytes, a human non-tumorigenic breast epithelial cell line (MCF10A), and epithelial Madin-Darby canine kidney (MDCK) cells. These cells were established from different lineages and are good models for investigating lineage specific variations of metal levels in vitro.

Primary myoblasts derived from mouse satellite cells constitute a well-suited in vitro model to investigate skeletal muscle differentiation. Proliferation of these cells is fast when cultured under high serum conditions. Differentiation into the muscular lineage is then induced by low serum conditions³⁰. The murine neuroblastoma (N2A) established cell line was derived from the mouse neural crest. These cells present neuronal and amoeboid stem cell morphology. Upon differentiation stimulus, the N2A cells present several properties of neurons, such as neurofilaments. N2A cells are used to investigate Alzheimer's disease, neurite outgrowth, and neurotoxicity³¹^,³²^,³³. The 3T3-L1 murine pre-adipocytes established cell line is commonly used to investigate the metabolic and physiological changes associated with adipogenesis. These cells present a fibroblast-like morphology, but once stimulated for differentiation, they present enzymatic activation associated with lipid synthesis and triglycerides accumulation. This can be observed as morphological changes to produce cytoplasmic lipid droplets³⁴^,³⁵. MCF10A is a non-tumor mammary epithelial cell line derived from a premenopausal woman with mammary fibrocystic disease³⁶. It has been widely used for biochemical, molecular, and cellular studies related to mammary carcinogenesis such as proliferation, cell migration, and invasion. The Madin-Darby canine kidney (MDCK) epithelial cell line has been extensively used to investigate the properties and molecular events associated with the establishment of the epithelial phenotype. Upon reaching confluence, these cells become polarized and establish cell-cell adhesions, characteristics of mammalian epithelial tissues³⁷.

To test the ability of AAS to measure the levels of Zn in mammalian cells, we analyzed whole and subcellular fractions (cytosol and nucleus) of these five cell lines. AAS measurements showed different concentrations of Zn in these cell types. Concentrations were lower in proliferating and differentiating primary myoblasts (4 to 7 nmol/mg of protein) and higher in the four established cell lines (ranging from 20 to 40 nmol/mg of protein). A small non-significant increase in Zn levels was detected in differentiating primary myoblasts and neuroblastoma cells when compared to proliferating cells. The opposite effect was detected in differentiated adipocytes. However, proliferating 3T3-L1 cells exhibited higher concentrations of the metal compared to differentiated cells. Importantly, in these three cell lines, subcellular fractionation showed that Zn is differentially distributed in the cytosol and nucleus according to the metabolic state of these cells. For instance, in proliferating myoblasts, N2A cells, and 3T3-L1 pre-adipocytes, a majority of the metal is localized to the nucleus. Upon induction of differentiation using specific cell treatments, Zn localized to the cytosol in these three cell types. Interestingly, both epithelial cell lines showed higher levels of Zn during proliferation compared to when reaching confluence, in which a characteristic tight monolayer was formed. In proliferating epithelial cells, the mammary cell line MCF10A had an equal Zn distribution between the cytosol and nucleus, while in the kidney-derived cell line, most of the metal was located in the nucleus. In these two cell types, when the cells reached confluence, Zn was predominantly located to the cytosol. These results demonstrate that GF-AAS is a highly sensitive and accurate technique for performing elemental analysis in low-yield samples. GF-AAS coupled with subcellular fractionation and can be adapted to investigate the levels of trace metal elements in different cell lines and tissues.

Protocol

1. Mammalian cell culture

General considerations
1. Follow the aseptic techniques for mammalian cell culture previously reviewed³⁸.
2. Maintain all cell lines in a humidified 5% CO₂ incubator at 37 °C. Cell culture conditions vary for each cell type. It is important to maintain appropriate culture conditions for each cell line used, as variations of these procedures will lead to aberrant phenotypes and failure of the cell culture.
3. Ensure acquaintance with the cell line of interest, and follow the protocols provided for each cell type selected for specific experiments (see some examples below).
General procedure for trypsinization and passing of adherent cells
1. Remove the cell culture media from the culture plate by aspiration.
2. Wash the cells gently using PBS without calcium and magnesium (5 mL per 10 cm² culture surface area). Add the wash solution to the side of the plate to avoid detaching cell monolayer; swirl the plate to maximize coverage of the solution. For epithelial cells, it is necessary to incubate the cells for 10 to 15 min in the incubator at 37 °C in PBS to facilitate the dissociation of cells in the following steps.
3. Remove the washing solution by aspiration.
4. Add enough dissociation reagent (0.25% trypsin) to the plate (approximately 0.7 mL per 10 cm²). Gently swirl the plate to get complete coverage of the cell monolayer.
5. Incubate the culture at room temperature for 2 to 5 min. The actual incubation time varies with the cell line used, in the case of epithelial cells is recommended to incubate for 10 to 15 min in the incubator at 37 °C. Observe the cells under the microscope for cell detachment.
6. When most of the cells have detached, recover the cell suspension in 5 mL of the pre-warmed plating or growth medium (see below for some examples). Mechanically dissociate cell clumps by pipetting over the cell monolayer several times.
7. Transfer the cell suspension to a 15 mL conical tube and centrifuge them at 1,000 x g for 2 to 5 min. The centrifugation speed and time vary based on the cell type. Remove the media by gentle aspiration, being careful not to touch the cell pellet. Resuspend the cell pellet in 5 to 10 mL of the appropriate pre-warmed growth medium and remove a sample for counting using a hemocytometer or an automatic cell counter.
8. Utilize the appropriate volume of the cell suspension to seed the recommended density for each specific cell line (see below for some examples). Supplement with the adequate volume of culture media according to the size of the culture dish and place the cells back into the incubator.
Primary myoblasts derived from mouse satellite cells
1. Before starting, prepare collagen-coated plates for primary myoblast growth. A solution containing 0.02% collagen, and 0.05 M acetic acid in deionized water must be sterilized by filtration with a 0.22 µm sterile filter device. Incubate the culture plates with the collagen solution overnight at 37 °C.
2. On the next day, aspirate the collagen solution, allow the plates to air dry in the sterile cabinet and store at 4 °C until use.
  NOTE: The minimal volumes of collagen are: for 35 mm = 1 mL; 60 mm = 2 mL; 10 cm = 5 mL.
3. Seed primary myoblasts at 1 x 10⁴ cells/cm² in 55 cm² collagen-coated culture plates. The proliferation media is Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 nutrient mixture culture media containing 20% fetal bovine serum (FBS), 25 ng/mL of recombinant basic fibroblastic growth factor (FGF), and 100 U/mL of penicillin/streptomycin.
  NOTE: Proliferating and stock cells should be maintained under 50% of confluency. Cell contact induces commitment of the cells to myogenic differentiation and impairs the growth capabilities of the culture.
4. For differentiating the primary myoblasts, allow the cells to reach 80% to 100% confluency, which normally occurs after 48 h of plating. Then, change to differentiation media (DMEM, 2% horse serum, 1% insulin, transferrin, selenium A supplement, and 100 U/mL of penicillin/streptomycin). Refresh the media daily until harvest.
Murine neuroblastoma (N2A)
1. Plate N2A cells at a density of 2 x 10⁴ cells/cm² in growth media, containing a mixture of reduced-serum medium/DMEM containing high glucose and L-glutamine (1:1, v:v), supplement with 10% FBS and 100 U/mL of penicillin/streptomycin. Maintain the stock culture under 50% confluency.
2. Induce differentiation of N2A cells once confluency reached 25% to 40%. Change to differentiation media containing a mixture of reduced-serum medium/DMEM, 1% FBS, and 20 µM retinoic acid for 7 to 10 days. Refresh the media every other day until cell harvest.
  NOTE: As N2A cells differentiate, some cells become round, detach easily, and may undergo apoptosis. Be careful when aspirating and adding the culture media to the plates.
3T3 L1 murine pre-adipocytes
1. Maintain mouse 3T3/L1 cells in DMEM with high glucose, supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. The adipogenic process is dependent in the confluence of the cells; therefore, do not let the stock culture grow over 50% confluency, as higher confluence will impair the ability of the cells to differentiate.
2. Plate the cells at 2 x 10⁴ cells/cm². At this density, the cells will reach confluence in 3 days.
3. Induce adipogenic differentiation two days after the cells reach 100% confluency. Induction media consists of DMEM containing 10% FBS, 10 μg/mL insulin, 0.5 mM 3-isobutyl-1-methyxanthine, 1 μM dexamethasone, and 10 μM troglitazone.
4. Replace the induction media after 48 h incubation to differentiation media (DMEM containing 10% FBS, and 5 μg/mL insulin). Refresh the media every other day until harvest. Representative samples of full differentiation are normally collected between 7 and 10 days after adipogenic induction.
  NOTE: As adipogenesis progresses, the cells become round and tend to detach easily. Be careful when aspirating and adding the culture media to the plates.
MCF10A cells
1. Plate MCF10A cells in DMEM/F12 (1:1, v:v), supplemented with 5% FBS, 100 U/mL penicillin/streptomycin, 0.5 µg/mL hydrocortisone, 10 µg/mL insulin, and 20 ng/mL epidermal growth factor (EGF). Recommended seeding density is 1 x 10⁵ cells/cm². Under these conditions, the cells will reach 100% confluency within 4 days. Refresh the media every 2 to 3 days until harvest.
  NOTE: MCF10A cells are difficult to detach. It is recommended to incubate the cells for 10 to 15 min at 37 °C in PBS free of calcium with 0.5 mM EDTA prior to trypsinization.
Madin-Darby canine kidney cells (MDCK)
1. Plate MDCK cells in DMEM supplemented with 10% FBS and 100 U/mL of penicillin/streptomycin. Recommended seeding density is 1 x 10⁵ cells/cm², where the cells will reach 100% confluency within 3 days. Refresh the media every 2 days until harvest.
  NOTE: MDCK cells are difficult to detach. It is recommended to incubate the cells for 5 to 10 min at 37 °C in PBS free of calcium and magnesium prior to trypsinization. To prevent cell clumping do not agitate the plates by hitting or shaking the flask while waiting for the cells to detach.

2. Mammalian cell culture sample preparation for AAS: whole cell and subcellular fractionation

Culture the cells of interest in 55 cm² plates. The use of smaller culture plates may yield samples with levels of metals under the detection limits of the GF-AAS.
Whole cell extract
1. Aspirate the culture media using a vacuum trap. Be sure to remove all traces of media. Rinse the cells from the desired time points or culture conditions three times with ice-cold PBS free of calcium and magnesium.
2. Immediately scrape the cells from the plate using a new plastic cell scraper in 1 mL of PBS free of calcium and magnesium. Transfer the sample to a 1.5 mL microcentrifuge tube and centrifuge for 2 min at 2,000 x g and aspirate the supernatant. The protocol can be paused here. Store pellet samples at -20 °C or proceed to step 2.4.
Subcellular fractionation
1. Aspirate the culture media using a vacuum trap. Be sure to remove all traces of media. Rinse the cells from the desired time points or culture conditions three times with ice-cold PBS free of calcium and magnesium. Scrape the cells off the plate with 1 mL of ice-cold PBS free of calcium and magnesium.
2. Transfer the samples to a 1.5 mL microcentrifuge tube and keep on ice. Centrifuge for 10 s at 10,000 x g. Continue rapid isolation of nuclei if metal analysis of cytoplasmic and nuclear fractions is desired. This procedure will minimize the potential loss of metals due to cell extraction^39,40.
3. Remove the supernatant by aspiration and resuspend the cell pellet in 400 μL of ice-cold PBS free of calcium and magnesium, containing 0.1% Nonidet P-40 (NP-40), a non-ionic detergent. Transfer 100 μL to a new microcentrifuge tube and consider it as the whole cell sample. This aliquot represents 25% of the sample.
4. Isolate nuclei by pipetting the cell suspension 5 to 10 times on ice with a P1000 micropipette tip. Nuclei isolation of epithelial cells requires a concentration of 0.5 % NP-40. Achieve cell dissociation by passing the cell suspension through a 26 ¾ G needle 10 to 15 times, followed by 10 to 15 passages through a 30 ½ G needle.
  NOTE: Verify the nuclei integrity by light microscopy using a 40x objective.
5. Centrifuge the remaining cell lysate suspension (300 μL) for 10 s at 10,000 x g. The supernatant will contain the cytosolic fraction. Transfer the 300 μL to a new microcentrifuge tube.
6. Rinse the pellet containing the nuclear fraction in 500 μL PBS free of calcium and magnesium, containing 0.1% NP-40, then centrifuge for 10 s at 10,000 x g. Remove the supernatant and resuspend the pellet containing the nuclei in 100 μL of the same solution.
  NOTE: The protocol can be paused here. Samples can be stored at -20 °C.
Lyse the cells by three sonication cycles (30 s on/30 s off, at 50 kHz, 200 W) for 5 min. Quantify total protein content in the sample by Bradford⁴¹.
Perform quality control of the purity of the fractions by western blot using antibodies specific for either the nuclear (histones, chromatin remodelers, nuclear matrix proteins) or cytosolic fractions (β-tubulin, β-actin) (Figure 2).
NOTE: Do not use sonicators with a metal tip. These can contaminate the sample with metals.

3. Zn content analysis of whole cell and subcellular fractions of mammalian cell cultures by atomic absorbance spectroscopy

Mineralize the cell culture samples (whole cell or nuclear fractions) by acid digestion using an equal volume of concentrated HNO₃ trace metal grade (CAUTION) for 1 h at 80 °C, then overnight at 20 °C.
Stop the reaction by adding 30% of the reaction volume of H₂O_2.Bring the sample volume to 500 μL with 18 MΩ purified water.
NOTE: HNO₃ handling should be done wearing chemical safety glasses, a face shield for splash protection, gloves, and an approved vapor respirator if adequate ventilation (fume hood) is not available. The protocol can be paused here. Samples can be stored at room temperature (RT).
Prepare standard solutions and experimental samples for Zn measurements by AAS equipped with a graphite furnace.
1. Use 2% (v/v) HNO₃solution in deionized water, coming from the same batch used for the standard curve as the blank solution for calibration throughout. Use analytical grade standards diluted in 18 MΩ purified water.
2. Prepare a standard of 1,000 ppb of Zn from a commercially available 1,000 ppm Zn stock solution with 2% (v/v) HNO₃ solution in deionized water. Prepare working standard solutions from the 100 ppb standard by diluting it to 5, 8, 10, 15, 20, and 25 ppb directly with 2% (v/v) HNO₃ solution in deionized water. Prepare the Zn standards and the blank with a 0.1% (1 g/L) Mg(NO₃)₂ matrix modifier.
  NOTE: The LOD of Zn is 0.01 ppb (µg/L). By increasing the concentration of the standards, the limit of linearity of Zn was determined to be 20 ppb (Figure 3).
3. Measure standard and samples under the same optimized conditions: flow rate of introduction of 250 mL/min, injection temperature of 20 °C, and GF temperature of 1,800 °C.
  NOTE: The GF temperature was increased to 2450 °C for cleanout steps, prior to the injection of a new sample or standard.
4. Optimize the lamp (C-HCL) to a current of 20 A, wavelength of 213.9 nm, and slit of 0.7 nm.
5. Measure the mineralized samples using the Zn standard curve to determine metal content. As small volumes are measured, the samples can evaporate if the measurements take long periods of time. Therefore, it is recommended to add water to the samples. Dilute the samples with 18 MΩ purified water treated with 0.01% HNO₃ (analytical grade) if the data obtained is beyond the LOD.
  NOTE: Typically, samples are diluted to a volume of 500 µL, placed in the automated autosampler of the AAS to be measured right after the standard curve is established. The required volume should be determined by the user and should consider final calculations of concentrations. Finishing the Zn determinations by AAS of a complete experiment in one attempt is recommended. Pausing the measurements may lead to large experimental variations and inconsistencies.
Convert the ppb measurements to molarity as obtained from Zn measured via AAS. Consider the mass of Zn ‎(65.39 amu). Divide the amount of ppb obtained by AAS by 63,390,000. In cells, the Zn concentrations range from units of pmol to µmol.
1. Divide the concentration of Zn (pmol or µmol) by the initial mass of protein in the sample determined by Bradford (step 2.4). This calculation will render the amount of metal (pmol or µmol Zn) contained per mg of protein of the sample.
  NOTE: It is important to consider the dilution factors utilized during the metal determinations.

Representative Results

We tested the ability of the GF-AAS to detect minute levels of Zn in mammalian cells (Figure 1). Thus, we cultured primary myoblasts derived from mouse satellite cells, and the established cell lines N2A (neuroblastoma derived), 3T3 L1 (adipocytes), MCF10A (breast epithelium), and MDCK cells (dog kidney epithelium). First, we isolated whole cell, cytosolic, and nuclear fractions of all these cell types and evaluated the purity of the fractions by western blot (Figure 2). Representative immunodetection of the chromatin remodeler Brg1 and tubulin as markers of nuclear and cytosolic fractions, respectively, demonstrated the suitability of the subcellular fractionation protocol described in section 2.3 to perform the elemental analysis (Figure 2). Whole cell and subcellular fractions were prepared as described above for GF-AAS analyses, and calibration of the equipment was performed to yield a typical linear standard curve (Figure 3).

Figure 4 shows representative light microscopy images of proliferating and differentiated or confluent monolayers of each cell type, and the corresponding Zn levels for these. Importantly, all the cell lines analyzed in this study showed Zn concentrations in the nM range. However, a differential distribution of the metal was detected. Differentiated primary myotubes exhibited higher levels of Zn than proliferating cells (Figure 4A). Notably, much of the ion was located to the nuclear fraction in both proliferating and differentiating myoblasts. A similar subcellular distribution of Zn was detected in the neuroblastoma derived cell line N2A (Figure 4B). However, measurements showed that N2A cells had Zn levels one order of magnitude higher than primary myoblasts. On the other hand, the established 3T3-L1 cell line exhibited higher levels of Zn when the pre-adipocytes were proliferating than when they were induced to differentiate and form mature adipocytes that accumulate lipids (Figure 4C).

We also measured Zn content in two different established epithelial cell lines: the MCF10A derived from human mammary gland (Figure 4D) and the dog kidney-derived MDCK (Figure 4E) cells. Interestingly, in both cases, higher levels of Zn in proliferating cells than in confluent monolayers were detected. However, the epithelial cells derived from the mammary gland showed metal levels that were 2-fold higher than that of the kidney cells. MCF10A cells showed equal levels of Zn between cytosolic and nuclear fractions in proliferating cells. Once MCF10A cells reach confluence, a 40% decrease in whole cell Zn levels was detected, and the metal was found to be most concentrated in the cytosolic fraction (Figure 4D). Conversely, proliferating MDCK cells exhibited higher levels of Zn in the nuclear fraction, compared to the cytosolic fraction, but when the MDCK cells reached confluence, a decrease of Zn in whole cells was detected, with the majority of this transition metal observed in the cytosolic fraction (Figure 4E).

Figure 1: Representative flow chart for elemental (Zn) analyses of mammalian cultured cells. Please click here to view a larger version of this figure.

Figure 2: Validation of the purity of fractions obtained via the rapid isolation of nuclei protocol³⁹^,⁴⁰. Representative western blot from differentiating primary myoblasts showing the purity of subcellular fractions. The chromatin remodeler enzyme Brg1 was used to identify the nuclear fraction, and tubulin was used to identify the cytosolic fraction. Please click here to view a larger version of this figure.

Figure 3: Calibration curve of Zn standards. Representative standard curve for Zn determined by GF-AAS. The GF-AAS was calibrated with the standard Zn solutions diluted at the following concentrations: 1, 3, 5, 7, 10, 15, 20, 25, and 30 ppb. Please click here to view a larger version of this figure.

Figure 4: Zinc levels in different mammalian cultured cells. Representative light micrographs and Zn content in whole cell extracts and cytosolic and nuclear fractions. Data represent the mean of three independent biological replicates ± SEM. (A) Murine primary myoblasts proliferating and differentiating for 2 days. (B) Murine neuroblastoma N2A cell line before and after induction to differentiate by retinoic acid treatment. (C) Murine 3T3-L1 pre-adipocytes and 6 days post differentiation. (D) Pre-confluent and confluent monolayer of the human mammary epithelial cell line MCF10A. (E) Pre-confluent and confluent monolayer of the kidney epithelial cell line MDCK. Data represent the average of three independent experiments ± SE. Student’s t-test shows significance, *p ≤ 0.05. Please click here to view a larger version of this figure.

Discussion

Atomic absorbance spectroscopy is a highly sensitive method for Zn quantification in small volume/mass biological samples. The described optimization of Zn measurement makes application of this method simple and guarantees ideal analytical conditions. Here, using GF-AAS, we determined the concentration of Zn in whole cells, and in cytosolic and nuclear fractions, from different cell lines. The results show that this technique renders comparable to those obtained by fluorescent probes and ICP-MS. For instance, several fluorescent probes showed labile mitochondrial Zn levels in the range of 0.1 to 300 pM, levels in the Golgi were below the picomolar range, and the endoplasmic reticulum ranged from 800 to 5,000 pM⁴², which are consistent with levels detected in the cytosolic fractions. Moreover, the Zn-responsive fluorophore FluoZin-3 showed accumulation of labile Zn in small cytosolic vesicles in MCF10A and in C2C12 cells⁴³^,⁴⁴, which are also consistent with the Zn quantified in the cytoplasm. Moreover, Zn analyses by ICP-MS performed by our group showed that whole cell levels Zn in differentiating primary myotubes⁴⁵ are similar to those obtained in this report by GF-AAS. In addition, examples of Zn determinations in C6 rat glioma cells by using AAS and Zinquin-E⁴⁶ have also rendered similar ranges to those observed in neuroblastoma N2A cells used here.

To date, diverse techniques have been developed to detect Zn in cells. However, GF-AAS remains an accurate and highly sensitive technique that not only allows detection of free Zn, but it also measures the metal complexed and bound to proteins and potentially to small molecules. Our group recently showed that measurements performed with cell-permeable fluorescent probe (which has a high affinity to bind free Zn²⁺) directly correlated with the levels of Zn detected by GF-AAS in proliferating and differentiating myoblasts⁴³. However, the results obtained by these probes may not be representative of whole cell or protein-bound Zn concentrations. Moreover, since Zn exists as a non-redox active species and as divalent cation, under physiological conditions, its detection in cellular systems remained a challenge. Novel Zn imaging techniques have become available, such as the state-of-the-art synchrotron-based X-ray fluorescence microscopy, radioisotopes, and laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS). Unfortunately, some of these techniques require resources inaccessible to the scientific community²⁹^,⁴²^,⁴⁷^,⁴⁸^,⁴⁹^,⁵⁰.

Studies have shown that ICP-MS is a more precise technique than AAS, as relative standard deviation values have been shown to be lower in ICP-MS determinations for metals like Zn. The likely explanation for this effect is that ICP-MS is intrinsically capable of eliminating chemical interferences, as the operating argon temperatures range from 5,000 to 10,000 °C. Some chemical bonds can be maintained at 3,000 °C, which is in the range for AAS operation. These bonds will be completely disrupted at above 6,000 °C. Therefore, these high temperatures reached in the plasma state by ICP-MS can eliminate chemical interferences, leading to better detection limits⁵¹. In addition, ICP-MS requires larger volume samples compared to GF-AAS, as the latter is capable of operating with volumes under 100 μL, which is significantly less than other spectroscopic methods such as AAS, ICP-OES, or ICP-MS. Given the small volumes involved in the method, GF-AAS is the ideal technique to determine Zn concentration in biological samples, primarily if the analysis involves subcellular fractions, metal detections in purified proteins, or small variations in cellular metal content due to mutations in transporters or metal binding proteins²⁹^,⁵². In addition, the sensitivity of the GF-AAS system to determine trace and ultra-trace concentrations (pg/mL to ng/mL) is another advantage.

Important considerations should be taken to ensure reliability of the GF-AAS data. These include adequate calibration, integrity of the graphite tube, and selection of suitable matrix modifier for electrothermal atomization. Matrix modifiers are chemical elements added to the sample, which affect the thermal processes taking place in the atomizer. These modifiers minimize the loss of analyte during pyrolysis and contribute to removal of matrix components. Overall, modifiers may change the sample matrix to evaporate the matrix components at lower temperatures and may work as analyte stabilizers. In addition to these considerations, it is important to perform adequate optimization of the steps for the atomizer temperature program.

Two main considerations should be taken, including 1) establishing the optimum pyrolysis temperature, which refers to the maximum temperature at the atomization step at which no analyte loss occurs, and which allows a maximum absorbance of the analyte with minimal background. One should also consider 2) establishing the optimum atomization temperature, which refers to the minimum temperature at which the analyte is fully evaporated and recorded as a reproducible signal or peak. One of the disadvantages of GF-AAS analyses is element interference, for which a thorough optimization and standardization are essential for accurate measurements. However, to date, GF-AAS represents an important tool in scientific research to detect the metal ions in diverse biological samples. Future applications of Zn (and other metals) detection methods by GF-AAS will include measuring the levels of metals in organs and tissues obtained from animal models or patient biopsies to better understand specific physiological needs for trace elements. In conclusion, GF-AAS is accurate, sensitive, cost-effective, and accessible, and this analytical technique will continue to improve as technological advances move forward for these detection systems.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Faculty Diversity Scholars Award from the University of Massachusetts Medical School to T.P.-B. N.N.-T. is supported by SEP-CONACYT, grant 279879. J.G.N is supported by the National Science Foundation Grant DBI 0959476. The authors are grateful to Dr. Daryl A. Bosco for providing the N2A cell line and to Daniella Cangussu for her technical support.

Materials

3-isobutyl-1-methylxanthine	Sigma Aldrich	I5879
Acetic Acid	Sigma Aldrich	1005706
Anti Brg1-antibody (G7)	Santa Cruz biotechnologies	sc-17796
Anti b-tubulin-antibody (BT7R)	Thermo Scientific	MA5-16308
Bradford	Biorad	5000205
Dexamethasone	Sigma Aldrich	D4902
Dulbecco's Modified Eagle's Media (DMEM)	ThermoFischer-Gibco	11965092
Dulbecco's Modified Eagle's Media/Nutrient Mix (DMEM/F12)	ThermoFischer-Gibco	11320033
Dulbecco's Phosphate Buffered Saline (DPBS)	ThermoFischer-Gibco	14190144
Epidemal Growth Factor (EGF)	Sigma Aldrich	E9644
Fetal Bovine Serum (FBS)	ThermoFischer-Gibco	16000044
Fibroblastic Growth Factor-Basic (FGF) (AA 10-155)	ThermoFischer-Gibco	PHG0024
Horse serum	ThermoFischer-Gibco	16050122
Hydrocortisone	Sigma Aldrich	H0888
Hydrogen Peroxide (H2O2)	Sigma Aldrich	95321
Insulin	Sigma Aldrich	91077C
Insulin-Transferrin-Selenium-A	ThermoFischer	51300044
Nitric Acid (HNO3)	Sigma Aldrich	438073
Nonidet P-40 (NP-40)	Thermo Scientific	85125
OptiMEM (Reduced Serum Media)	ThermoFischer-Gibco	31985070
Penicillin-Streptomycin	ThermoFischer-Gibco	15140148
PureCol (Collagen)	Advanced BioMatrix	5005
Retionic Acid	Sigma Aldrich	PHR1187
Troglitazone	Sigma Aldrich	648469-M
Trypsin-EDTA (0.25%), phenol red	ThermoFischer-Gibco	25200056
Zinc (Zn) Pure Single-Element Standard, 1,000 µg/mL, 2% HNO3	Perkin Elmer	N9300168
Established Cell Lines
3T3-L1	American Type Culture Collection	CL-173
MCDK	American Type Culture Collection	CCL-34
MCF10A	American Type Culture Collection	CRL-10317
N2A	American Type Culture Collection	CCL-131
Equipment
Atomic Absortion spectrophotometer	PerkinElmer	Aanalyst 800
Bioruptor	Diagnode	UCD-200

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Gordon, S. J., Xiao, Y., Paskavitz, A. L., Navarro-Tito, N., Navea, J. G., Padilla-Benavides, T. Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells. J. Vis. Exp. (147), e59519, doi:10.3791/59519 (2019).