RESUMO
BACKGROUND: The modern management of phenylketonuria (PKU) consists of generalized newborn screening (NBS) for hyperphenylalaninemia (HPA), confirmation of HPA in children detected in the NBS, introduction of dietary treatment in the first weeks of life, followed by monitoring the treatment of PKU for decades to maintain phenylalaninemia within the limits that will not affect the brain. The present study aimed to evaluate the usefulness of two chromatographic methodologies for determination of plasma Phe level in the routine management of PKU: the two dimensional thin layer chromatography (2D - TLC) and the high performance liquid chromatography (HPLC) procedures, respectively. MATERIAL AND METHODS: Samples of blood from 23 children with HPA detected by neonatal screening or with confirmed PKU who received treatment by low-Phe diet were analyzed to estimate the plasma Phe level by the two chromatographic procedures. RESULTS: In case of three subjects the very low concentrations of plasma Phe could not be detected by the 2D - TLC methodology, since the spot was not visible on the chromatogram. In four patients the differences between the values of plasma Phe determined by the two methodologies are not statistically significant, while in fifteen subjects the differences are highly statistically significant. This is due to the greater errors that appear in the case of 2D - TLC methodology. In the range of concentrations of plasma Phe higher than 360 µmol/L (which is the cut-off value for HPA), although in four cases there were statistically significant differences in the level of plasma Phe determined by the two methodologies, the value obtained by the 2D - TLC methodology was high enough to influence the decision of changing the diet so that HPA is kept under control. In addition, the intense spot of Phe on the 2D - TLC chromatogram may be detected even by un unexperienced laboratory specialist. CONCLUSION: The HPLC procedure for measurement of plasma Phe level is very suitable to be used in the routine management of PKU. The 2D - TLC procedure may be accompanied by relatively high errors; however, it detects patients with severe PKU.
RESUMO
OBJECTIVE: To compare two chromatographic methodologies for determination of plasma phenylalanine (Phe) and their usefulness for diagnosing hyperphenylalaninemia (HPA) and phenylketonuria (PKU). METHODS: The plasma amino acids were isolated and concentrated from blood collected from infants with HPA detected by newborn screening. The plasma Phe was determined in parallel by HPLC and by image-densitometry of 2D-TLC plates. RESULTS: Typical examples of 2D-TLC plates and HPLC chromatograms from infants with HPA and PKU are presented and evaluated. The Phe spot was visible on 2D - TLC plates at Phe concentrations higher than 300 µmol/L. The standard calibration curve traced after image-densitometry of the Phe spot presented high dispersion of values at each concentration of Phe, high SD values, the equation of the curve having a low R-squared value (0.862). In contrast, the standard calibration curve obtained by HPLC shows linearity on the range of concentrations from 100 - 16,000 µmol/L, extremely small SD values, the equation of the curve has a very high R-squared value (0.999). CONCLUSIONS: The HPLC methodology is appropriate to confirm HPA detected by newborn or selective screening of PKU. The 2D - TLC methodology is adequate to detect patients with severe PKU.
RESUMO
When compared with values recorded in 14 control subjects, the 15 overweight patients with type 2 diabetes displayed significantly increased activities of serum alanineaminotransferase (172% of mean values in controls), gamma-glutamyltransferase (253%) and cholinesterase (139%). A much wider dispersion of individual values for the two firstly mentioned enzymes was however noted so that their correlation with serum triglycerides levels were weaker (r = 0.373; p < 0.05 and r = 0.451; p < 0.05 respectively) than the same correlation obtained for serum cholinesterase (r = 0.760; p < 0.001). In two other studies including 28 controls and 30 diabetic patients serum cholinesterase was found to be significantly correlated with serum levels of insulin (r = 0.622; p < 0.001), C-peptide (r = 0.652; p < 0.001) and free fatty acid (r = 0.821; p < 0.001). Circumstantial evidence is provided that insulin resistance and an increased flux of free fatty acids from adipose tissue to the liver would stimulate the hepatic synthesis of serum cholinesterase.