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.
ABSTRACT
PURPOSE: The purpose is to determine the effect of food on the bioavailability of S-1, an oral formulation of the 5-fluorouracil (5FU) prodrug Ftorafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP), a dihydropyrimidine dehydrogenase inhibitor, and oxonic acid (an inhibitor of 5FU phosphoribosylation in normal gut mucosa) in a molar ratio of 1:0.4:1. EXPERIMENTAL DESIGN: Eighteen patients received a single dose of S-1 of 35 mg/m(2) with (535-885 kcal) or without food in a crossover study design: in arm A without breakfast on day -7 and with breakfast on day 0 and in arm B the reversed sequence. Blood samples were taken before and after S-1 administration. This food effect was evaluated according to the Food and Drug Administration guidelines using log-transformed data. RESULTS: Pharmacokinetic parameters for 5FU without breakfast were as follows: Tmax, 107 min; Cmax, 1.60 microm; area under the plasma concentration-time curve (AUC) 441 microm x min; and T(1/2), 104 min. Fasting decreased Tmax of FT, 5FU, CDHP, and oxonic acid significantly (P < 0.006) and increased the Cmax (P < 0.013). The food/fast ratio for the AUC of FT was not different, which for 5FU was 0.84 (P = 0.041), for CDHP was 0.89 (P = 0.191), for oxonic acid was 0.48 (P < 0.0005), and for cyanuric acid, the breakdown product of oxonic acid, was 5.1 (P = 0.019). Accumulation of uracil, indicative for dihydropyrimidine dehydrogenase inhibition, was not affected, as well as the T(1/2) of FT, 5FU, CDHP, and oxonic acid. Evaluation of the log-transformed data demonstrated that the 90% confidence interval for the food/fast ratio for the Cmax and AUC of FT, 5FU, CDHP, and uracil were within 70-143% and 80-125%, respectively, indicating no food effect. Only for oxonic acid and cyanuric acid were these values outside this interval. CONCLUSIONS: Food intake affected only the pharmacokinetics of the S-1 constituent oxonic acid but not of FT, CDHP, and 5FU. Because oxonic acid is included to protect against gastrointestinal toxicity, this observation might affect the gastrointestinal toxicity and thus the efficacy of S-1.
