Radiolabeling Heme and Porphyrin with ¹⁴C-Glycine or ¹⁴C δ-Aminolevulinic Acid

Abstract

Radiolabeling enables the quantitation of newly synthesized heme and porphyrin, allowing us to distinguish heme synthesis rates from total cellular heme. Here, we describe a protocol for labeling heme with ¹⁴C-glycine or ALA and the sequential extraction of heme and porphyrin from the same samples for quantitation by liquid scintillation.

1. Introduction

Radiolabeling techniques to quantitate de novo heme and porphyrin synthesis have played an essential role in advancing studies of iron metabolism. Even with current advances in mass spectrometry-based metabolomics techniques, the extraction and detection of porphyrins in a mix of cellular metabolites remains challenging for several reasons. First, porphyrins are light-sensitive, requiring handling under limited lighting conditions. Second, heme and porphyrin are miscible in lipid and are solubilized under similar conditions as lipids, which are vastly more abundant in the cell; the low concentration of heme/porphyrin in comparison to other metabolites causes numerous challenges in mass spectrometric detection. Non-isotopic methods such as HPLC provide a static snapshot of total cellular heme/porphyrin rather than a dynamic view of heme metabolism. In combination with non-isotopic quantitation methods and enzymatic assays, radiolabeling techniques provide insight into the dynamics of substrate utilization in regulation of heme synthesis.

Newly synthesized heme is typically labeled with ⁵⁵Fe [1, 2], ¹⁴C-glycine, or ¹⁴C-aminolevulinate (ALA) [3-5]. The advantage of ¹⁴C glycine and ALA labeling is the ability to simultaneously quantify free protoporphyrin within the same experiment. In particular, labeling with ¹⁴C-ALA standardizes the amount of ALA entering the labeled heme population, thus allowing one to isolate changes in the heme synthesis pathway that occur downstream of ALAS.

This protocol is a modification of a method initially described by Ponka and Schulman [5]. Briefly, cultured cells are glycine-starved in minimal essential media (MEM) for an hour prior to labeling. ¹⁴C-glycine or ¹⁴C-ALA labeling is carried out in MEM supplemented with dialyzed FBS using holo-transferrin as an iron source. One may also use Fe-PIH or other iron chelates as a source of iron during labeling as previously described [5, 6]. Cells are lysed in RIPA buffer containing Triton X-100, and radiolabeled heme and porphyrins are extracted in ethylacetate. Subsequently, heme is isolated by sequentially washing the ethylacetate extract with sodium acetate and HCl. Protoporphyrin is obtained by washing the porphyrin fraction with saturated sodium acetate and HCl. Labeled heme and protoporphyrin are quantitated by liquid scintillation.

2. Materials

2.1. Cell Culture

1. Murine erythroleukemia and 3 T3 cells should be cultured in complete DMEM media.

   • DMEM (Gibco 11,995–065) supplemented with.

     1. 1% penicillin/streptomycin (Gibco 15140-122).
     2. 1% L-glutamine (Gibco 25031-081).
     3. 10% fetal bovine serum (FBS) (R&D Biosystems S11550).

2. Differentiation is carried out in total DMEM media supplemented with 2% DMSO (Corning MT25950CQC).

3. G1E-ER4 cells should be cultured in the following media as described [7].

   • IMDM (Gibco 12440-046) supplemented with.

     • 15% fetal bovine serum (FBS) (GeminiBio 100-525).
     • 1.25% penicillin/streptomycin (Gibco 15140-122).
     • 125 uM monothioglycerol (MTG) (MP Biomedicals 155723).
     • 50 ng/mL stem cell factor (SCF) (455-MC/CF).
     • 2 U/mL erythropoietin (EPO) (PHC9631).

4. Differentiation is carried out by adding 1 × 10⁻⁸ mM estradiol (Sigma E2758-1g).

2.2. Labeling Reagents

• Minimum essential media (MEM), Gibco #12360038.

• Labeling media-MEM media supplemented with dialyzed FBS, 10% final concentration (Sigma F0392).

• Holo-transferrin (10 mg/mL). Holo-transferrin powder can be obtained from Sigma (T4132). Dissolve all the powder in the vial with sterile water (cell culture grade) to a concentration of 10 mg/mL. Store in 200 uL aliquots at −20 °C.

• ¹⁴C-glycine (Perkin Elmer or American Radiochemicals), stock concentration 0.1 uCi/uL or ¹⁴C-ALA (American Radiochemicals), stock concentration 0.1 uCi/uL.

2.3. Heme and Porphyrin Extraction

Prepare all reagents using reverse osmosis water.

• RIPA buffer (store at 4 °C).

  150 mM NaCl.

  50 mM Tris pH 8.0.

  1% NP40.

  1% Triton X-100.

  0.5% sodium deoxycholate.

• Protein assay dye reagent concentrate (Biorad #5000006).

• Bovine serum albumin stock (10 mg/mL) for protein quantification.

• Ethylacetate:acetic acid (3:1).

• 3% sodium acetate.

• 1.5 N HCl.

• Saturated sodium acetate.

  Note: Dissolve at 50 °C and then cool to room temperature before using to ensure true saturation.

• 0.1 N HCl.

• Scintillation fluid (Ultima Gold, Perkin Elmer #6013329).

3. Methods

3.1. Cell Culture

1. MEL cells are seeded at a density of approximately 50 k cells/mL and grown in 50 mL complete DMEM media in 50 mL until the concentration is about 1.5 million cells/mL, as determined with a hemacytometer. To differentiate MEL cells, seed undifferentiated MEL cell samples at a cell concentration of 3 × 10⁴cells/mL in 50 mL complete DMEM media (1.5 million cells) for 72 h to obtain actively growing cultures. To differentiate, seed these cells at a concentration of 3 × 10⁵cells/ml (15 million cells) in 50 mL media with 2% DMSO for 72 h. Cell density and DMSO concentration (ranging from 1.5% to 2%) can vary between labs, so these are conditions that should be optimized.

2. G1E-ER4 cells were grown in complete IMDM media in 100 mL media until the concentration reaches 1–1.5 million cells/mL. To differentiate, undifferentiated G1E-ER4 cell samples were set up with 7.5 million cells in 50 mL media and left to expand for 48 h. G1E-ER4 cells were differentiated at a concentration of 7 × 10⁵cells/mL (35 million cells) in 50 mL media with 1 × 10⁻⁸ mM estradiol for 48 h.

3. For both cell lines, 40 million cells should be used for each sample on the day of labeling.

4. Adherent cells (we have used 3 T3 cells; adapt growth conditions to cell type of choice) should be actively growing and about 80% confluent on the day of labeling. 6 wells of cells grown in a 6-well plate should be used for each sample. 3 T3 cells are grown in complete DMEM media.

3.2. Labeling and Heme/Porphyrin Extraction

1. 1 h before labeling, remove growth media from cells and glycine-starve cells with pre-warmed MEM media, which contains no glycine. 1 well of 3 T3 cells in 6-well plates are starved in 1 mL MEM. For MEL/G1E-ER4 cells, spin down 40 million cells and resuspend in 10 mL MEM. Leave cells in a CO₂ incubator at 37 °C.

2. 3 T3 cells: Remove MEM media. Replace with 2 mL labeling media per well. Add 15 uL of holotransferrin (10 mg/mL) into each well. Incubate for 30 min at 37 °C. A full 6-well plate is 2 technical replicates, with 3 wells per technical replicate. MEL/G1E-ER4 cells: Spin down and aspirate MEM media. Resuspend 40 million cells in 4 mL of labeling media and dispense into 2 wells of a 12-well plate. Add 15 uL of holo-transferrin (10 mg/mL) into each well. Incubate for 30 min at 37 °C.

3. Add 1.5 uCi (15 uL) of ¹⁴C-glycine or ¹⁴C-ALA to each well. When choosing which label to use, see Note 1– ALA is generally less efficiently taken up by the cells resulting in lower signal.

4. Incubate for 2 h at 37 °C.

5. Washing: Adherent cells: aspirate labeling media and wash once with 5 mL ice cold PBS. Erythroid suspension cells: spin down cells in each well in a 2 mL Eppendorf tube at 2000 g for 5 min. Wash once in 2 mL ice cold PBS. Resuspend well, gently vortex. Spin down at 2000 g for 5 min and remove the PBS.

6. Cell lysis: Adherent cells: Add 500 uL ice cold RIPA buffer in one well. Resuspend cells, pipet into second and third well. 3 wells of cells are used for 1 technical replicate. You will end up with 2 tubes of 500 uL lysates from a 6-well plate. Erythroid suspension cells: Resuspend each cell pellet in 500 uL of ice cold RIPA buffer.

   Note: At this point, you can freeze the cell lysates at −80 °C and continue subsequent steps later. We do not recommend freezing the cell pellet and resuspending at a later stage—when we did this, we found that the results were less consistent even across technical replicates.

7. Protein quantitation: Keep 5 uL of cell lysate for protein quantitation with the Bradford assay. Make a standard curve with dilutions of 10 mg/mL BSA stock for comparison.

8. Extraction of total heme/porphyrin:

   1. Add 500 uL ethylacetate: acetic acid (3:1) to the lysate and vortex for 1 full minute, then centrifuge at maximum speed for 5 min.
      Notes: Sufficient vortexing is extremely important to reliably extract heme and porphyrin from the lysate. Insufficient vortexing will result in an inconsistent and weak signal. Some protocols call for centrifuging in a 4 °C centrifuge. In our experience, this is unnecessary.
   2. Transfer 350–400 uL of the top layer into a 1.5 uL centrifuge tube. This is the heme/porphyrin fraction.
      Note: When carrying out the extraction in differentiated erythroid cells, the top layer should be pink and the bottom layer should be colorless. This is an indication that the heme has been correctly extracted.
   3. Repeat extraction from the cell lysate—500 uL ethylacetate:acetic acid (3:1)—to the lysate and vortex for 1 full minute and then centrifuge at maximum speed for 5 min.
   4. Pool 300–350 uL of the top layer with the tube from (b).

9. Wash the pooled ethylacetate layers with 3% sodium acetate. This is done by adding 750 uL of 3% sodium acetate to the pooled ethylacetate layers, vortexing for 30 s, and centrifuging at maximum speed for 5 min.

   Notes: The interface between layers may be blurred when centrifugation is carried out at room temperature due to heat generation, making it difficult to distinguish between the layers. Simply remove the tubes from the centrifuge and let them cool. The interface will become clear when the tubes have cooled to room temperature.

10. Separation of heme and porphyrin:

    1. Transfer 300 uL of the top layer to a new tube.
    2. Add 600 uLof 1.5 N HCl.
    3. Vortex for 1 min and centrifuge at maximum speed for 5 min.
       Notes: If centrifugation is done at room temperature, let the tubes cool after centrifugation to see the interface clearly. The top ethylacetate layer from this step is heme; the bottom layer contains porphyrins.

11. Quantitation of heme: Transfer 200 uL of the top layer to a scintillation vial. If using differentiated erythroid cells, the top layer may be pink from heme (this can be an internal quality check for the extraction) if the cells are well-differentiated. Add 10 mL of scintillation fluid and count in a scintillation counter using the ¹⁴C program.

12. Porphyrin extraction:

    1. Split the bottom layer into 2 tubes (i.e., 1 sample is split into 2 tubes). This is approximately 350 ul in each tube.
    2. To each tube, add 500 uL of saturated sodium acetate and 500 uL of ethylacetate:acetic acid (3:1).
    3. Vortex for 1 min.
    4. Centrifuge at maximum speed for 5 min.
    5. The top layer contains coproporphyrin and protoporphyrin.
13. Protoporphyrin extraction:

    1. Transfer 300 ul of the top layer to a new tube. Pool the top layers from the same sample together (total 600 ul)
    2. Wash with 600 ul 0.1 N HCl and vortex for 30 s.
       Note: This step eliminates the coproporphyrins.
    3. Centrifuge at max speed for 5 min.
    4. Transfer 600 uL of the top layer to a scintillation vial.
       Note: This extract contains protoporphyrins, which consist of protoporphyrin IX and auto-oxidized protoporphyrinogen IX.
    5. Add 10 mL of scintillation fluid and count using the 14C program.
    6. For analysis, normalize heme and porphyrin radioactive counts (CPM or DPM) to protein concentration. (Refer to Note 2 on normalization controls and Note 3 on omitting the decolorization steps described in the original protocol (Ref 5).

4. Notes

1. We obtained lower counts with ¹⁴C-ALA as compared to ¹⁴C-glycine used as a label, despite the ALA being committed to heme synthesis, while glycine is used in many other processes such as protein synthesis. We speculate that this discrepancy reflects the relative uptake of the labeled molecules into the cell, as glycine (although a nonessential amino acid) is frequently obtained from the diet or extracellular environment and has specific cellular transporters [5, 8], while most ALA is synthesized from glycine and succinyl CoA and transported within the cell, not from the extracellular environment.

2. While it is common to normalize the data to protein concentration, it is sometimes advisable to normalize to cell number. This is particularly true when there is a concern that treatments (such as erythroid differentiation) or genetic modifications (such as defects in erythroid differentiation that cause hemoglobin deficiencies—a major component of protein content in red cells) have altered total protein content within the cells.

3. The original protocol described by Ponka and Schulman called for decolorizing heme extracts with H₂O₂ as the color of heme can quench the signal in liquid scintillation. For the numbers of erythroid cells used in this protocol, it has not been necessary, but it may be required when labeling larger numbers of cells or reticulocytes as previously described.