Shu-Chu Shiesh (Presenter)
National Cheng Kung University
Authorship: Shu-Chu Shiesh (1), Kuan-Ying Li (1), Ching-Hsuan Yang (1), Yu-Chen Su (1), Chiung-Yu Chen (2)
(1) Department of Medical Laboratory Science and Biotechnology; (2) Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| Short Abstract Neonatal intrahepatic cholestasis caused by citrin deficiency, is characterized by galactosemia, hyperlipidemia, hyperammonemia, and multiple aminoacidemia. Galactose-free formulas are effective for resolving clinical symptoms. This study aimed to investigate the impacts of substitution of 18 mM glucose in culture medium by 9 mM glucose/galactose, 13.5 mM glucose or 9 mM glucose on cellular arginine, dimethylarginine and oxidative stress in primary hepatocyte. The measurement of arginine and metabolites was performed using API 5000 tandem mass spectrometer with positive ionization mode. Oxidative stress increased in hepatocytes cultured in glucose-insufficient medium, compared to 18 mM glucose medium. Intracellular concentrations of arginine and lysine did not change, but citrulline, ADMA, and SDMA increased in AML12 cultured with glucose insufficient medium. |
Long Abstract
Argininosuccinate synthetase (ASS, EC 6.3.4.5), catalyzing the condensation of aspartate and citrulline to argininosuccinate, is a rate-limiting enzyme in the synthesis of nitric oxide (NO) and urea. In cells, citrulline is a byproduct in citrulline-NO cycle. Through the reactions of ASS and argininosuccinate lyase, citrulline is recycled and converted to arginine. Adult-onset type II citrullinemia (CTLN2), which is caused by the deficiency of citrin, a calcium-stimulated mitochondrial aspartate-glutamate carrier, is characterized by hypoglycemia, galactosemia, hyperlipidemia, hyperammonemia, and multiple aminoacidemia, such as citrullinemia, and argininemia. Low-carbohydrate diets are effective for CTLN2 to lowering the accumulation of cytosolic NADH and hypertriglyceridemia. Although serum arginine concentration increased in CTLN2 patients, the supplemented arginine is of help for the formation of urea. Carbohydrate counting (diets containing carbohydrates, proteins and lipids with a ratio of 45¡V10¡V45%), was effective for the reduction of serum ammonia levels. Therefore, this study aimed to investigate the impacts of substitution of glucose (18 mM) in culture medium by 9 mM glucose/galactose, 13.5 mM glucose or low glucose (9 mM) on cellular concentrations of arginine and dimethylarginine and oxidative stress in primary mouse hepatocyte AML12. The measurement of arginine and dimethylated arginine was performed using API 5000 tandem mass spectrometer with positive electrospray ionization mode. The transitions (m/z) were 175.1 to 60.0 for arginine, 203.1 to 70.1 for asymmetry dimethylarginine (ADMA), 203.3 to 172.1 for symmetry dimethylarginine (SDMA), 176.3 to 159.2 for citrulline, and 147.1 to 130.2 for lysine. Oxidative stress increased in hepatocytes cultured in glucose-insufficient medium, compared to 18 mM glucose medium. Citrulline concentrations were increased in AML12 cells cultured with 9 mM glucose + 9 mM galactose, or 9 mM glucose-containing medium, compared to 18 mM glucose containing medium (0.56 and 0.68 v.s. 0.24 µmol/g). Intracellular concentrations of arginine and lysine did not change, but ADMA, and SDMA increased in AML12 cultured with glucose insufficient medium. SDMA concentrations were found to be increased at a dose-dependent manner in AML12 cells cultured in medium containing 18 mM, 13.5 mM, or 9 mM glucose. Taken together, glucose insufficiency increased oxidative stress with ADMA and SDMA accumulation, and further causes the increase of citrulline. The increase of ADMA may decrease NO synthesis in hepatocytes.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |