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. Author manuscript; available in PMC: 2025 Nov 26.
Published in final edited form as: Methods Enzymol. 2025 Feb 26;715:257–270. doi: 10.1016/bs.mie.2025.01.071

Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples

Chad R Schultz a,b, Elizabeth A VanSickle b,c, Caleb P Bupp a,b,c, André S Bachmann a,b,*
PMCID: PMC12646276  NIHMSID: NIHMS2124621  PMID: 40382142

Abstract

Polyamines are aliphatic molecules that include putrescine, spermidine, and spermine. Polyamines are present in most living organisms including humans. These positively charged molecules play important roles in cell physiology and pathology by contributing to embryonic cell development, regulation of cell division and, if overproduced, the stimulation of cancer cell proliferation and tumorigenesis. We recently discovered Bachmann-Bupp Syndrome (BABS); a rare neurodevelopmental disorder linked to de novo mutations in the ornithine decarboxylase 1 (ODC1) gene. ODC1 gene mutations that are linked to BABS always produce C-terminally truncated versions of the enzyme ornithine decarboxylase (ODC). These shortened ODC proteins remain enzymatically active and are not cleared by the proteasome, therefore leading to ODC protein accumulation in cells. ODC is a key enzyme of polyamine biosynthesis by converting ornithine to putrescine, and if accumulated, can lead to high putrescine levels in human cells including red blood cells (RBCs) and primary dermal fibroblasts.

Here we describe how to quantitatively measure ODC enzymatic activity and the polyamines by a radiolabeled 14C-ornithine assay and by reverse phase (RP)-HPLC, respectively. While these methods have been developed decades ago, many publications provide incomplete protocols with omission of experimental details, which inadvertently can lead to mistakes, inconclusive results, and failed experiments. There is a growing number of laboratories that have become interested in exploring polyamines (in part due to metabolomics analyses in human health-related studies). The detailed protocols of this chapter provide step-by-step guidance detailing how to measure ODC activity and polyamines in human RBCs.

1. Introduction

Polyamines were discovered several centuries ago, in the year 1677 by Antonie van Leeuwenhoek (Lewenhoeck, 1677). They are polycationic molecules that interact with negatively charged molecules including DNA, RNA, proteins, and lipids. Polyamines are ubiquitous and found in almost all organisms in both prokaryotes and eukaryotes including bacteria, parasites, plants, and mammals. In humans, polyamines have been primarily studied in the context of cancer and infectious disease (Bachmann & Geerts, 2018; Casero & Marton, 2007; Casero, Murray Stewart, & Pegg, 2018; Gerner & Meyskens, 2004; Holbert, Cullen, Casero, & Stewart, 2022). Most prominently, polyamines and treatments were explored for West African sleeping sickness (trypanosomiasis) (Alirol et al., 2013; Alirol et al., 2012; Bacchi et al., 1983; Priotto et al., 2009) and several types of cancer including colorectal and prostate cancer (Gerner & Meyskens, 2004; Meyskens et al., 2008; Raj et al., 2013; Simoneau, Gerner, Phung, McLaren, & Meyskens, 2001) as well as the pediatric cancer neuroblastoma (Bachmann, Geerts, & Sholler, 2012; Geerts et al., 2010; Hogarty et al., 2024; Oesterheld et al., 2023; Saulnier Sholler et al., 2015; Sholler et al., 2018; Wallick et al., 2005). The irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO, also known as Eflornithine) received FDA approval in 1990 (intravenous formulation) to treat trypanosomiasis (in combination with nifurtimox). In 2000, the FDA approved a topical formulation that includes 13.9 % DFMO to treat hirsutism (Wolf et al., 2007). In 2023, the FDA approved DFMO (oral formulation, available as tablets) now known as Iwilfin for the treatment and relapse prevention of adult and pediatric patients with high-risk neuroblastoma. In addition, many other diseases are currently being studied to understand the importance of polyamines in pathophysiology and to explore opportunities to treat such diseases with specific polyamine pathway inhibitors (Bachmann & Levin, 2012).

In 2018, we discovered a neurodevelopmental disorder referred to as Bachmann-Bupp Syndrome (BABS) that is linked to C-terminally truncated forms of ODC (Bupp, Schultz, Uhl, Rajasekaran, & Bachmann, 2018). BABS was the second polyamine disorder, or polyaminopathy, to be identified in the polyamine pathway, after Snyder-Robinson Syndrome (SRS) (Arena et al., 1996; Cason et al., 2003; Schwartz, Peron, & Kutler, 2020; Snyder & Robinson, 1969). We repurposed DFMO to treat BABS patients (Bachmann, VanSickle, Michael, Vipond, & Bupp, 2024). We showed that untreated BABS patients have excessive baseline amounts of accumulated ODC due to the lack of proper clearance via the proteasome, leading to higher ODC enzymatic activity and high levels of putrescine in human red blood cells (RBCs) and primary dermal fibroblasts (Schultz, Bupp, Rajasekaran, & Bachmann, 2019). Treatment of the first BABS patients with DFMO rapidly reduced N1-acetylputrescine levels in patient plasma compared to levels before treatment and led to remarkable overall patient phenotype improvements, including hair regrowth and developmental progress (Rajasekaran et al., 2021). Currently, a total of 17 BABS patients have been identified worldwide and six BABS patients are being treated with DFMO, leading to similar patient responses (unpublished data).

2. Rationale

Although ODC activity assays and polyamine analyses are described in many prior publications, these protocols often omit important experimental details which inadvertently can lead to mistakes, inconclusive results, and failed experiments. Moreover, most ODC and polyamine determinations are made using cell lines and not human biological samples. Given the importance of polyamines and their recent association with a plethora of new biological processes, human diseases, genetic disorders, and various medical conditions (sometimes identified “by accident” via broad spectrum metabolomics analyses), we are publishing our detailed protocols to measure ODC enzymatic activity and polyamines in human RBCs. The blood was collected from patients with BABS or healthy controls and RBCs prepared as described in a separate chapter of this book (see VanSickle et al.).

3. Experimental section

3.1. RBC preparation

3.1.1. Equipment

  • Handheld homogenizer (VWR, Radnor PA, USA)

  • Refrigerated Microcentrifuge (Eppendorf, Hamburg, Germany)

3.1.2. Reagents

  • Liquid nitrogen

  • 1.8 ml microcentrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA)

  • ODC Breaking Buffer – 25 mM Tris HCL, 0.1 mM EDTA, 2.5 mM DTT

3.1.3. Procedure

  1. Pipet 50 μl of each RBC sample into a microcentrifuge tube.

  2. Add 200 μl of ODC Breaking Buffer to the RBCs.

  3. Snap freeze the mixture with liquid nitrogen and allow the sample to thaw on ice.

  4. Homogenize the mixture for 10–15 s

  5. Centrifuge the samples for 10 min at 12,000 rpm.

  6. Pipet the supernatant into a fresh microcentrifuge tube to use for polyamine preparations and ODC activity assay.

3.1.4. Notes

  1. DTT should be added fresh (day of) to the ODC breaking buffer.

  2. All of the steps of the procedure should be done on ice.

  3. The RBC samples are a thick slurry, so it is helpful to cut the end of the pipet tip in order to draw up the 50 μl of sample.

3.2. Protein quantification assay

3.2.1. Equipment

  • CLARIOstar Plate Reader (BMG Labtech, Ortenberg, Germany)

3.2.2. Reagents

  • Costar Assay Plate, 96-well (#9017 Corning, Corning, NY)

  • Albumin Standard, 2 mg/ml (#23209 Thermo Fisher Scientific, Waltham, MA)

  • Protein Assay Dye Concentrate (#5000006, Bio-Rad Laboratories, Hercules, CA)

3.2.3. Procedure

  1. Dilute the albumin standard from 2 mg/ml to 100 μg/ml (1:20) using deionized water.

  2. Setup a standard curve in the top row of a 96-well plate using the diluted albumin standard as follows (Table 1):

  3. For each RBC preparation sample pipet into a 96-well plate well 48 μl of water plus 2 μl of sample.

  4. Add 200 μl of 1:3 diluted Protein Assay Dye Concentrate to all the wells containing the albumin standard or RBC preparation and incubate at room temperature for 10 min.

  5. Measure the absorbance value at OD595 on the CLARIOstar plate reader.

  6. Using Microscoft Excel, plot the standard curve with the concentration of albumin on the Y-axis and the OD595 readings on the X-axis.

  7. Use the formula for the standard curve line to determine the unknown concentrations of the RBC preparations.

  8. Based on the determined concentration calculate the mg of protein in 50 μl of each RBC preparation.

Table 1.

Diluted albumin standards for protein quantification assay.

100 μg/ml Albumin (μl) Water (μl) [Albumin] μg/ml
0 0 0
3.13 46.87 6.25
6.25 43.75 12.5
12.5 37.5 25
25 25 50
50 0 100
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3.2.4. Notes

  1. Each concentration of the standard curve as well as each RBC preparation sample should be done at least in duplicate.

  2. The RBC preparations are typically very high in protein concentration, so you may have to dilute the preparations 1:5 or 1:10 prior to using in the protein quantification assay so that your OD595 reading will be within the value range of the standard curve.

  3. The amount of protein (mg) in 50 μl of sample will be used to normalize the ODC activity values and polyamine amounts across samples.

3.3. ODC activity assay

3.3.1. Equipment

  • Isotemp 37 °C shaking waterbath (Fisher Scientific, Waltham, MA)

  • TriCarb 4910 TR Liquid Scintillation Counter (Perkin Elmer, Waltham, MA)

3.3.2. Reagents

  • 0.5 M Tris – HCl pH 7.5

  • 2 mM pyridoxal – 5 – phosphate

  • 25 mM DTT

  • 20 mM L-ornithine

  • 55 mCi/mmol L- [1–14C] ornithine (American Radiolabeled Chemicals, St. Louis, MO)

  • 0.1 N sodium hydroxide

  • 5 M sulfuric acid

  • 1.8 ml microcentrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA)

  • Beckman PolyQ scintillation vials (#566350, Fisher Scientific, Waltham MA)

  • Circular Filter Paper, 20 mm diameter (#1001-020, Cytiva Life Sciences, Marlborough, MA)

  • Research Products International Bio-Safe II complete counting cocktail (#111195, Fisher Scientific, Waltham, MA)

3.3.3. Procedure

  1. Prepare the ODC activity assay reaction buffer mix as follows:
    1. Per reaction:
      1. 2.5 μl of 0.5 M Tris-HCl pH 7.5
      2. 1 μl 20 mM L-ornithine
      3. 5 μl of 2 mM pyridoxal – 5- phosphate
      4. 12.5 μl of 25 mM DTT
      5. 1 μl L-[1-14C] ornithine (55 mCi/mmol)
      6. 178 μl deionized water
    2. Allow the mix to sit for 30 min at room temperature.

  2. Pipet 50 μl of RBC preparation samples from section one into a 1.8 ml microcentrifuge tube.

  3. For each sample prepare a Beckman PolyQ scintillation vial with a 20 mm circular piece of filter paper soaked with 200 μl of 0.1 N sodium hydroxide to capture the 14C labeled carbon dioxide produced in the reaction.

  4. Add 200 μl of ODC activity assay reaction buffer to the 50 μl of RBC preparation sample and place the 1.8 ml microcentrifuge tube with the cap open into the prepared scintillation vial.

  5. Place the cap on the vial and incubate the samples in the 37 °C water bath while shaking at 30 rpm for 2 h.

  6. Remove the samples from the water bath and add 250 μl of 5 M sulfuric acid to the reaction mix to end the reaction.

  7. Incubate the samples for an additional 30 min in the 37 °C water bath while shaking at 30 rpm.

  8. Remove the reaction mix from the microcentrifuge tube with a 1 ml pipet and put it into a liquid radiation waste container.

  9. Using forceps, grab the opened cap of the microcentrifuge tube and remove it from the scintillation vial and place it in a solid radiation waste container.

  10. Add 5 ml of Research Products International Bio-Safe II complete counting cocktail to each vial.

  11. Cap the vial and centrifuge briefly (2–3 s) and allow the vials to sit overnight.

  12. Run the vials in the TriCarb 4910 TR Liquid Scintillation Counter, capturing the disintegrations per minute (DPM) and microCurie (μCi) for 1 min per sample.

  13. Use the original activity of the L-[1-14C] ornithine of 55 mCi/mmol to convert the μCi values to molar values of CO2 liberated in the reaction.

  14. ODC activity values are reported as pmol CO2/120 min/mg protein (Fig. 1A).

Fig. 1.

Fig. 1

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(A) Ornithine decarboxylase (ODC) enzyme activity measurement in red blood cells (RBCs) using the 14C-ornithine radiolabeled assay. ODC activity in Bachmann-Bupp Syndrome (BABS) RBCs was significantly higher compared to control RBCs. (B) Polyamine (PA) concentration of putrescine (Put), spermidine (Spd), and spermine (Spm) measured by reverse phase (RP)-HPLC indicating a significant increase in putrescine levels in BABS RBCs compared to control RBCs. (C) Chromatogram showing the peaks of internal PA standards that are used on every RP-HPLC run. (D) Chromatogram showing PA peaks in a BABS RBC sample. Numbers represent the retention time of each metabolite. To enhance the visibility of the PA peaks, the scale was reduced, which caused the standard peak to be cut off.

3.3.4. Notes

  1. Samples are run in duplicate in the ODC activity assay.

  2. Aliquots of 2 mM pyridoxal-5-phosphate, 25 mM DTT, and 20 mM L-ornithine are made in advance and kept frozen at −20 °C. Aliquots are used only once.

3.4. Dansylating polyamines in the RBC preparations

3.4.1. Equipment

  • Refrigerated Microcentrifuge (Eppendorf, Hamburg, Germany)

  • Microcentrifuge Tube Thermomixer R (Eppendorf, Hamburg, Germany)

3.4.2. Reagents

  • 1.8 ml microcentrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA)

  • Perchloric acid buffer (0.2 M perchloric acid/1 M sodium chloride)

  • 0.15 mM 1,7 diaminoheptane

  • 1 M sodium carbonate

  • 5 mg/ml dansyl choride dissolved in acetone

  • 1 M proline

  • 4 ml amber glass vials with cap (C4015-17AW, Thermo Fisher Scientific, Waltham, MA)

  • Methylene chloride

  • Methanol

  • Hypersep C18, 25 mg cartridges (#60108-376, Thermo Fisher Scientific, Waltham, MA)

  • 2 ml glass vials w/septa for autosampler (#C5000-180, Thermo Fisher Scientific, Waltham, MA)

3.4.3. Procedure

  1. Pipet 50 μl of each RBC preparation sample into a 1.8 ml microcentrifuge tube.

  2. Add 100 μl of perchloric acid buffer to each sample and vortex briefly. This will protonate the polyamines and precipitate out the protein in the sample.

  3. Pellet the precipitated protein by spinning the samples at 12,000 rpm for 10 min at room temperature using the microcentrifuge.

  4. Transfer the supernatant to a clean 1.8 ml microcentrifuge tube.

  5. Add 30 μl of 0.15 mM 1,7 diaminoheptane internal standard to each sample.

  6. Add 200 μl of 1 M sodium carbonate to each sample.

  7. Add 400 μl of 5 mg/ml dansyl chloride in acetone to each sample and vortex for a few seconds.

  8. Incubate the samples at 37 °C while shaking at 300 rpm in the thermomixer R for 1 h.

  9. Add 100 μl of 1 M proline to each sample.

  10. Incubate the samples at 37 °C while shaking at 300 rpm in the thermomixer R for 20 min.

  11. Transfer the samples to the 4 ml amber glass vials with caps.

  12. In a fume hood add 1 ml of methylene chloride to each sample and mix by inverting ten times. This will extract out the dansylated polyamines into the clear bottom layer.

  13. Using a 1 ml pipet tip, go to the bottom of the vial and extract as much of the clear bottom layer without drawing up the upper milky white layer and transfer it to a clean 4 ml amber vial.

  14. Leave the cap off of the vial with the extracted polyamines and allow the methylene chloride to evaporate overnight in the fume hood.

  15. Add 1 ml of methanol to each sample and vortex to reconstitute the polyamines.

  16. To preclear the samples so that they do not cause a clog in the HPLC system, the samples are passed through Hypersep C18 25 mg cartridges. Pipet the 1 ml of sample onto the cartridge. Using a hose attached to a lab air line, use the air to push the sample through the cartridge into a clean uncapped 2 ml glass vial that will be used on the HPLC autosampler.

  17. Screw the cap with septa onto the 2 ml glass vials and load onto the autosampler trays to run on the HPLC.

3.4.4. Notes

  1. Each RBC preparation sample should be processed in duplicate.

  2. Accurate pipetting of the 1,7 diaminoheptane standard is crucial as the relative molar response of the polyamine peaks to the 1,7 diaminoheptane standard peak will be used to quantify the amount of polyamines in a sample.

  3. Step 15 above can be shortened if there is access to a vacufuge to evaporate the methylene chloride.

3.5. Running samples on the HPLC

3.5.1. Equipment

  1. Dionex Ultimate HPLC System with an autosampler and fluorescence detector (Thermo Fisher Scientific, Waltham, MA)

  2. Syncronis C18 (250 × 4.6 mm, 5 μm particle size) reverse phase (RP) column (Thermo Fisher Scientific, Waltham, MA)

3.5.2. Reagents

  1. Buffer A: 10 mM Sodium −1- heptane sulfonate in 10% acetonitrile, pH 3.5

  2. Buffer B: 100% Acetonitrile

3.5.3. HPLC running conditions

  1. Set the flow rate to 0.7 ml per minute.

  2. Equilibrate the column with 55% buffer B/45% buffer A for 7 min prior to injecting the first sample.

  3. Inject 20 μl of dansylated polyamine sample into the HPLC system.

  4. Program the buffer gradient to elute the polyamines as follows:
    1. Isocratic solution of 55% buffer B/45% buffer A for 28 min.
    2. Slowly ramp to 95% buffer B/5% buffer A for 20 min. Hold this gradient for 20 min to elute the spermine peak.
    3. Drop back to 55% buffer B/45% buffer A for 7 min.
    4. Re-equilibrate the column in 55% buffer B/45% buffer A for 7 min prior to the next sample being injected.
  5. The approximate elution times using the above gradients are as follows (Fig. 1B–D):
    1. Putrescine: 40 min
    2. 1,7 diaminoheptane internal standard: 54 min
    3. Spermidine: 59 min
    4. Spermine: 66 min

3.5.4. HPLC analysis

  1. For each HPLC system, a relative molar response between each of the polyamines and the 1,7 diaminoheptane internal standard must be determined. This is done by dansylating equal amounts of each individual polyamine with an equal amount of 1,7 diaminoheptane and determining the peak area ratio for each polyamine in comparison to the internal standard.

  2. Determine the polyamine concentration in a sample by measuring the peak areas for each polyamine and 1,7 diaminoheptane standard. Then use the relative molar response determined above to determine the number of moles of each polyamine and normalize to the amount of protein in each sample. Polyamine (putrescine, spermidine, spermine) concentration values are reported as nmol PA/mg protein (Fig. 1B).

3.5.5. Notes

  1. The run time for each sample is over 1 h, so the use of an autosampler is highly recommended to allow for samples to be run when personnel is not in the lab.

  2. Variations in the retention time of the polyamine peaks can occur if the acetonitrile percentage in Buffer A differs slightly between runs. It is important to include a polyamines standard sample to each run to ensure proper identification of each peak.

Acknowledgments

We acknowledge the National Institutes of Health grant award R01 HD110500 (A.S.B., C.P.B.) for the provided funds to study BABS and SRS and we thank the MSU-Spectrum Health Alliance Corporation (SH-MSU-ACF RG101298) for the provided support to generate the data shown in this book chapter. We also thank the late Dr. Patrick Woster (Medical University of South Carolina, Charleston, South Carolina) for his countless contributions to the polyamine field and for providing us and the polyamine community with DFMO over many years.

Footnotes

Competing interests

A.S.B. and C.P.B. are listed inventors of two U.S. patents (US 11,273,137 B2 and US 12,194,010 B2) issued on March 15, 2022 and January 14, 2025, respectively, entitled “Methods and compositions to prevent and treat disorders associated with mutations in the ODC1 gene” and Michigan State University and Corewell Health have an exclusive licensing agreement with Orbus Therapeutics, Inc. C.P.B. provides consulting services for Orbus Therapeutics. A.S.B. is sole inventor of a U.S. patent (US 9,072,778) issued on July 7, 2015, entitled “Treatment regimen for N-Myc, C-Myc, and L-Myc amplified and overexpressed tumors”. No potential conflicts of interest were disclosed by the other authors.

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