Fructose-1,6-bisphosphatase (F1,6BPase) is an enzyme expressed predominantly in liver and kidney and is one of the rate-limiting enzymes of hepatic gluconeogenesis. The enzyme catalyzes the hydrolysis of Fructose-1,6-biphosphate (F1,6BP) to Fructose-6-phosphate (F6P) and inorganic phospate. In diabetes, the lack of insulin (type 1) or resistance (type 2) to insulin alters the gene expression pattern in the liver, such that F1,6BPase is upregulated. F1,6BPase inhibitors reduce hepatic glucose output and lower blood glucose by inhibiting the elevated rate of gluconeogenesis present in diabetic patients, and represent interesting compounds for the development of type 2 diabetes new drugs. Standard spectrophotometric assays measure F1,6BPase ability to hydrolyze the inorganic phosphate from F1,6BP. The phosphate released is quantified spectrophotometrically at 620 nm as a complex with Ammonium Molybdate and Malachite Green1. Although this enzymatic assay is simple and fast, the method has the disadvantage of being laborious and, moreover, gives an indirect measure of the enzyme activity. The two sugar phosphates are especially difficult to analyze due to their polarity, structural similarity, and non characteristic UV absorption. LC-MS analysis (in negative polarity) in TIC and SIM mode was performed as an alternative specific and sensitive method for the simultaneous analysis of F1,6BP and F6P and for enzyme activity determination. Chromatographic separation of F1,6BP and F6P was obtained on a Luna NH2 column (150x2 mm I.D.) with the following mobile phase: triethylamine acetate buffer (5 mM)/acetonitrile (80/20) (v/v) in a binary pH gradient (from pH=9 to pH=10 in 15 minutes) at the flow rate of 0.3 mL/min. The SIM mode showed the pseudo-molecolar ion [M-H]- of F6P at m/z= 259, while the pseudo-molecolar ion [M-H]- of F1,6BP was detected at m/z= 339. Characterization of the sugar phosphates was achieved through ESI-MS-MS analysis, which revealed the main product ion for F1,6BP at 241 m/z (originated from H2PO4- loss), while the H2PO4- fragment itself was detected at 96.9 m/z for F6P. Furthermore, LC-MS analysis allowed to eliminate the interferences derived from the salts component of the enzyme catalysis buffer. This method was applied for routine sample analysis of the enzymatic reaction and for inhibition studies.

LC-ESI-MS-MS METHOD FOR OFFLINE FRUCTOSE-1,6-BISPHOSPHATASE ACTIVITY DETERMINATION.

MANCINI, FRANCESCA;FIORI, JESSICA;CAVRINI, VANNI;ANDRISANO, VINCENZA
2004

Abstract

Fructose-1,6-bisphosphatase (F1,6BPase) is an enzyme expressed predominantly in liver and kidney and is one of the rate-limiting enzymes of hepatic gluconeogenesis. The enzyme catalyzes the hydrolysis of Fructose-1,6-biphosphate (F1,6BP) to Fructose-6-phosphate (F6P) and inorganic phospate. In diabetes, the lack of insulin (type 1) or resistance (type 2) to insulin alters the gene expression pattern in the liver, such that F1,6BPase is upregulated. F1,6BPase inhibitors reduce hepatic glucose output and lower blood glucose by inhibiting the elevated rate of gluconeogenesis present in diabetic patients, and represent interesting compounds for the development of type 2 diabetes new drugs. Standard spectrophotometric assays measure F1,6BPase ability to hydrolyze the inorganic phosphate from F1,6BP. The phosphate released is quantified spectrophotometrically at 620 nm as a complex with Ammonium Molybdate and Malachite Green1. Although this enzymatic assay is simple and fast, the method has the disadvantage of being laborious and, moreover, gives an indirect measure of the enzyme activity. The two sugar phosphates are especially difficult to analyze due to their polarity, structural similarity, and non characteristic UV absorption. LC-MS analysis (in negative polarity) in TIC and SIM mode was performed as an alternative specific and sensitive method for the simultaneous analysis of F1,6BP and F6P and for enzyme activity determination. Chromatographic separation of F1,6BP and F6P was obtained on a Luna NH2 column (150x2 mm I.D.) with the following mobile phase: triethylamine acetate buffer (5 mM)/acetonitrile (80/20) (v/v) in a binary pH gradient (from pH=9 to pH=10 in 15 minutes) at the flow rate of 0.3 mL/min. The SIM mode showed the pseudo-molecolar ion [M-H]- of F6P at m/z= 259, while the pseudo-molecolar ion [M-H]- of F1,6BP was detected at m/z= 339. Characterization of the sugar phosphates was achieved through ESI-MS-MS analysis, which revealed the main product ion for F1,6BP at 241 m/z (originated from H2PO4- loss), while the H2PO4- fragment itself was detected at 96.9 m/z for F6P. Furthermore, LC-MS analysis allowed to eliminate the interferences derived from the salts component of the enzyme catalysis buffer. This method was applied for routine sample analysis of the enzymatic reaction and for inhibition studies.
15th International Symposium on Pharmaceutical and Biomedical Analysis, PBA2004
296
296
F. Mancini; J. Fiori; V. Cavrini; V. Andrisano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/13418
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