Systemic availability of guanidinoacetate affects GABAA receptor function and seizure threshold in GAMT deficient mice.

Deficiency of guanidinoacetate methyltransferase (GAMT) causes creatine depletion and guanidinoacetate accumulation in brain with the latter deemed to be responsible for the severe seizure disorder seen in affected patients. We studied electrical brain activity and GABAA mediated mechanisms of B6J.Cg-Gamt(tm1Isb) mice. Electrocorticographic (ECoG) monitoring of pharmacological treatments with ornithine (5 % in drinking water for 5-18 days) and/or Picrotoxin (PTX) (a GABAA receptor antagonist) (1.5 mg/kg, I.P.) in Gamt(MUT) and Gamt(WT) groups [n = 3, mean age (SEM) = 6.9 (0.2) weeks]. Mice were fitted with two frontal and two parietal epidural electrodes under ketamine/xylazine anesthesia. Baseline and test recordings were performed for determination of seizure activity over a 2 h period. The ECoG baseline of Gamt(MUT) exhibited an abnormal monotonous cortical rhythm (7-8 Hz) with little variability during awake and sleep states compared to wild type recordings. Ornithine treatment and also PTX administration led to a relative normalization of the Gamt(MUT) ECoG phenotype. Gamt(WT) on PTX exhibited electro-behavioral seizures, whereas the Gamt(MUT) did not have PTX induced seizures at the same PTX dose. Gamt(MUT) treated with both ornithine and PTX did not show electro-behavioral seizures while ornithine elevated the PTX seizure threshold of Gamt(MUT) mice even further. These data demonstrate: (1) that there is expression of electrical seizure activity in this Gamt-deficient transgenic mouse strain, and (2) that the systemic availability of guanidinoacetate affects GABAA receptor function and seizure thresholds. These findings are directly and clinically relevant for patients with a creatine-deficiency syndrome due to genetic defects in GAMT and provide a rational basis for a combined ornithine/picrotoxin therapeutic intervention.

Stable isotope dilution microquantification of creatine metabolites in plasma, whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency.

BACKGROUND: To develop an accurate stable isotope dilution assay for simultaneous quantification of creatine metabolites ornithine, arginine, creatine, creatinine, and guanidinoacetate in very small blood sample volumes to study creatine metabolism in mice. METHODS: Liquid-chromatography (C18) tandem mass spectrometry with butylation was performed in positive ionization mode. Stable isotope dilution assay with external calibration was applied to three different specimen types, plasma, whole blood and dried blood spot (DBS). RESULTS: Analytical separation, sensitivity, accuracy, and linearity of the assay were adequate. The stable isotope dilution assay in plasma revealed no significant bias to gold standard methods for the respective analytes. Compared to plasma, we observed an overestimate of creatine and creatinine (2- to 5-fold and 1.2- to 2-fold, respectively) in whole-blood and DBS, and an underestimate of arginine (2.5-fold) in DBS. Validation of the assay in mouse models of creatine deficiency revealed plasma creatine metabolite pattern in good accordance with those observed in human GAMT and AGAT deficiency. Single dose intraperitoneal application of ornithine in wild-type mice lead to fast ornithine uptake (Tmax ≤ 10 min) and elimination (T1/2=24 min), and a decline of guanidinoacetate. CONCLUSION: The assay is fast and reliable to study creatine metabolism and pharmacokinetics in mouse models of creatine deficiency.

Pyruvate dehydrogenase phosphatase 1 (PDP1) null mutation produces a lethal infantile phenotype.

Pyruvate dehydrogenase phosphatase deficiency has previously only been confirmed at the molecular level in two brothers and two breeds of dog with exercise intolerance. A female patient, who died at 6 months, presented with lactic acidemia in the neonatal period with serum lactate levels ranging from 2.5 to 17 mM. Failure of dichloroacetate to activate the PDH complex in skin fibroblasts was evident, but not in early passages. A homozygous c.277G > T (p.E93X) nonsense mutation in the PDP1 gene was identified in genomic DNA and immunoblotting showed a complete absence of PDP1 protein in mitochondria. Native PDHC activity could be restored by the addition of either recombinant PDP1 or PDP2. This highlights the role of PDP2, the second phosphatase isoform, in PDP1-deficient patients for the first time. We conclude that the severity of the clinical course associated with PDP1 deficiency can be quite variable depending on the exact nature of the molecular defect.

Multiple acyl-CoA-dehydrogenase deficiency (MADD): use of acylcarnitines and fatty acids to monitor the response to dietary treatment.

The treatment of multiple acyl-CoA-dehydrogenase deficiency (MADD) includes a low-fat, low-protein, high-carbohydrate diet, avoiding long fasting periods. However, there is no useful biochemical marker to determine the response to different diets or fasting periods. The aims of this study are to report a patient with MADD, diagnosed through a newborn screening program using tandem mass spectrometry, to assess her response to different feedings, and to evaluate the usefulness of acylcarnitines and FFA to monitor the response to dietary changes. The patient was diagnosed at 6 d. Family history revealed three dead siblings. Five tests were performed, one with breast milk and the subsequent four after giving the patient a bottle of a low-fat, low-protein formula (F), F with glucose polymers (GP), F+GP plus uncooked corn starch (CS), or F+GP+CS preceded by amylase. The results showed that acylcarnitines, FFA, and total nonesterified fatty acids levels were greatly improved at 2 and 4 h on F+GP compared with breast milk. At 6 mo of age, the test with F+CS was repeated to assess the response to a longer fast. The results were similar at 2 and 4 h, but showed a marked increase of acylcarnitines, FFA, and total nonesterified fatty acids at 6 h. The increase of these metabolites could not be avoided by the use of F+GP+CS, but was prevented when amylase was used simultaneously. The patient is currently 3.9 y old and has normal growth and development. We conclude that diagnosis of MADD through a newborn screening program using tandem mass spectrometry is suitable; acylcarnitines and FFA are useful to monitor the response to treatment; and exogenous amylase allows the use of CS in small children with MADD. This therapeutic approach may be an alternative to the use of continuous overnight feedings used for young children with severe fatty acid oxidation defects. Early diagnosis and treatment may change the natural history of MADD.