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

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.