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Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.

ABSTRACT: INTRODUCTION:Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of metabolism (IEM), clinically characterized by intellectual disability, developmental delay, seizures, and movement disorders. Biochemical diagnosis of GAMT deficiency is based on the measurement of creatine and guanidinoacetate in urine, plasma, or CSF and is confirmed genetically by DNA analysis or by enzyme assay in lymphoblasts or fibroblasts. To obtain enough cells, these cells need to be cultured for at least 1 month. A less time-consuming diagnostic functional test is needed, since GAMT deficiency is a candidate for newborn screening (NBS) programs, to be able to confirm or rule out this IEM after an initial positive result in the NBS. METHODS:Stable-isotope-labeled 13C2-guanidinoacetate and 2H3-S-adenosylmethionine (SAM) were used, which are converted by GAMT present in lymphocyte extracts into 2H3-13C2-creatine. The formed 2H3-13C2-creatine was butylated and subsequently measured by liquid chromatography tandem mass-spectrometry (LC-MS/MS). RESULTS:We measured GAMT enzyme activity in lymphocyte extracts of 24 controls, 3 GAMT deficient patients and of 2 parents proven to be carrier. Because GAMT activity decreases when isolation time after venipuncture increases, reference values were obtained for 2 control groups: isolation on the day of venipuncture (27-130 pmol/h/mg) and 1 day afterwards (15-146 pmol/h/mg). Deficient patients had no detectable GAMT activity. The two carriers had GAMT activity within the normal range. CONCLUSION:We designed a fast, less invasive, and valid method to measure GAMT activity in lymphocytes using LC-MS/MS analysis without the need of time-consuming and laborious cell culture.

SUBMITTER: Berends LM

PROVIDER: S-EPMC5740046 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.

Berends Lisette M LM Struys Eduard A EA Roos Birthe B Holwerda Ulbe U Jansen Erwin E W EEW Salomons Gajja S GS Wamelink Mirjam M C MMC

JIMD reports 20170221

<h4>Introduction</h4>Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of metabolism (IEM), clinically characterized by intellectual disability, developmental delay, seizures, and movement disorders. Biochemical diagnosis of GAMT deficiency is based on the measurement of creatine and guanidinoacetate in urine, plasma, or CSF and is confirmed genetically by DNA analysis or by enzyme assay in lymphoblasts or fibroblasts. To obtain enough cells, these cells need to be cultured ...[more]

PMID: 28220408

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Project description:BackgroundGuanidinoacetate (GAA) induces epileptogenesis and neurotoxicity in the brain. As epileptic animal models have been reported to show elevated cerebral GAA levels, the processing mechanism of GAA in the brain is important for maintaining brain homeostasis. We have revealed that GAA in the cerebrospinal fluid (CSF) is removed by incorporation into the choroid plexus epithelial cells (CPxEpic), which form the blood-CSF barrier (BCSFB). However, the processing mechanism of GAA incorporated into CPxEpic remains unknown. We have reported that monocarboxylate transporter 12 (MCT12) functions as an efflux transporter of GAA and creatine, a metabolite of GAA, in the kidneys and liver. Therefore, we aimed to clarify the role of MCT12 in GAA dynamics in CPxEpic.MethodsProtein expression and localization in CPxEpic were evaluated using immunohistochemistry. Metabolic analysis was performed using high-performance liquid chromatography (HPLC) 24 h after the addition of [14C]GAA to TR-CSFB3 cells, which are conditionally immortalized rat CPxEpic. The efflux transport of [14C]creatine was evaluated in TR-CSFB3 cells after transfection with MCT12 small interfering RNA (siRNA). The CSF-to-brain parenchyma transfer of creatine was measured after intracerebroventricular injection in rats.ResultsImmunohistochemical staining revealed that MCT12-derived signals merged with those of the marker protein at the apical membrane of CPxEpic, suggesting that MCT12 is localized on the apical membrane of CPxEpic. The expression levels of guanidinoacetate N-methyltransferase (GAMT), which catalyzes the conversion of GAA to creatine, in TR-CSFB3 cells was also indicated, and GAA was considered to be metabolized to creatine after influx transport into CPxEpic, after which creatine was released into the CSF. Creatine release from TR-CSFB3 cells decreased following MCT12 knockdown. The contribution ratio of MCT12 to the release of creatine was more than 50%. The clearance of CSF-to-brain parenchyma transfer of creatine was 4.65 µL/(min·g brain), suggesting that biosynthesized creatine in CPxEpic is released into the CSF and supplied to the brain parenchyma.ConclusionsIn CPxEpic, GAA is metabolized to creatine via GAMT. Biosynthesized creatine is then released into the CSF via MCT12 and supplied to the brain parenchyma.

Dataset Information

Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.

Publications

Guanidinoacetate Methyltransferase Activity in Lymphocytes, for a Fast Diagnosis.

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