HPLC assay for guanidinoacetate methyltransferase

Abstract: Guanidinoacetate methyltransferase (GAMT; EC2.1.1.2),1 the enzyme catalyzing the Wnal step in creatine biosynthesis by transferring a methyl group from Sadenosylmethionine to guanidinoacetic acid (GAA), has been considered to be highly expressed in a few organs such as kidney, liver, and pancreas [1]. Low activity is reported in skeletal and cardiac muscle, mouse neuroblastoma cells, and human fetal lung Wbroblasts [2]. The discovery of the Wrst case of GAMT deWciency [3], and more recently of other patients with the same enzymatic defect, has drawn attention to the analytical procedures used to measure the activity of this enzyme in human cells with low expression so as to avoid the more invasive hepatic biopsy. Previously published assays for GAMT activity [2,4,5] used radiolabeled molecules as substrates to reach high sensitivity and speciWcity. Moreover, all of these methods consist of numerous steps (e.g., extraction, puriWcation of the extracts, quantiWcation), require high amounts of cells, and show high unspeciWc blank radioactivity and, therefore, are more useful for detecting GAMT in tissues than in cells with low activity [2]. Ilas et al. [6] developed a speciWc and sensitive method to measure GAMT activity in human-cultured Wbroblasts, Epstein–Barr virus-transformed lymphoblasts, and cultivated amniotic cells but still with radiolabeled substrate. In their method, creatine is separated from [14C]guanidinoacetate in HPLC and is quantiWed in a beta counter. In this article, we present a nonradioactive method for measuring GAMT activity in lymphoblasts based on the quantiWcation of creatine, the end product of the GAMT, by using HPLC with uv detector.

Link to article: https://www.sciencedirect.com/science/article/abs/pii/S000326970400418X?via%3Dihub

PubMed: https://pubmed.ncbi.nlm.nih.gov/15246013/

Authors: Maria Grazia Alessandrı̀, Lisa Celati, Roberta Battini, Fulvia Baldinotti, Chike Item, Vincenzo Leuzzi, Giovanni Cioni

Key Terms: GAMT, Diagnostic, In vitro, Basic Science