Determination of phenylalanine and tyrosine in dried blood specimens by ion-exchange chromatography using the Hitachi L-8800 analyzer.

OBJECTIVES: The treatment for phenylketonuria (PKU) includes monitoring blood phenylalanine (Phe) levels on a regular basis. To reduce inconvenience to the patient and family, blood specimens on filter paper can be obtained at home and mailed to the clinic or analytical laboratory. For this reason, we validated an 8-min isothermal and isocratic HPLC method using the Hitachi L-8800 analyzer for quantitation of Phe and tyrosine (Tyr) from dried blood specimens (DBS). DESIGN AND METHODS: The method was worked out using DBS fortified with Phe and Tyr. For method comparison, blood samples from 31 PKU patients and 5 non-PKU volunteers were analyzed as DBS by HPLC using the Hitachi L-8800 analyzer, and compared both to plasma analyzed by HPLC and DBS analyzed using tandem mass spectrometry (MS/MS). RESULTS: For HPLC analysis of DBS, the within-run precision for Phe and Tyr was < or = 5.1% and < or = 4.5%, respectively, and total precision measured over a 3-month period was < or = 7.2% and < or = 8.7%, respectively. Correlation analysis was performed using results from fresh plasma analyzed by HPLC (r = 0.988 for Phe, r = 0.964 for Tyr) and from DBS analyzed by MS/MS (r = 0.960 for Phe, r = 0.942 for Tyr). Difference plots revealed good agreement between the HPLC and MS/MS methods. CONCLUSIONS: Determination of Phe and Tyr in DBS using this HPLC technique compares well with other methods. This technique with its short analytical time is convenient for monitoring patients with PKU and might be particularly useful in centers following many patients.

Infantile hypermethioninemia and hyperhomocysteinemia due to high methionine intake: a diagnostic trap.

Studies were carried out to identify the cause of combined severe hypermethioninemia and moderate hyperhomocysteinemia in a cluster of 10 infants ascertained between 1999 and early 2001. Although several were thought initially to have cystathionine beta-synthase (CBS) deficiency and treated accordingly, CBS deficiency and other known genetic causes of hypermethioninemia were ruled out by assay of CBS activity in fibroblasts of four patients and by assays of plasma cystathionine and S-adenosylmethionine. Retrospective data on dietary methionine intakes and plasma concentrations of methionine and related metabolites established that the hypermethioninemia in nine of the 10 babies was related to ingestion of an infant protein hydrolysate formula, the methionine content of which had been increased from May 1998 to February 2001. The formula in question has now been reformulated and is no longer available. The 10th infant manifested similar metabolic abnormalities while receiving TPN containing excessive methionine. Brain MRI abnormalities indicative of cerebral edema, most marked in the cerebral cortex and posterior brainstem, occurred in two patients near times of extreme hypermethioninemia. Metabolic and MRI abnormalities resolved when the methionine intake decreased. A third infant had a normal MRI 1 day after the formula was changed. The possible relationship between extreme hypermethioninemia and cerebral edema is discussed and a working hypothesis offered to explain the relative sensitivity of the inferior colliculi, based upon the facts that this is the region most active in glucose utilization and that Na(+),K(+)-ATPase is inhibited by methionine and related metabolites.