sodium-dodecyl-sulfate and Phenylketonurias

The urinary metabolic marker compounds, namely phenylpyruvic acid (PPA), 2-hydroxyphenylacetic acid (oOPAA), 4-hydroxyphenylacetic acid (pOPAA), phenyllactic acid (PLA) and phenylacetic acid (PAA) of phenylketonuric individuals were detected by a novel method of micellar electrokinetic capillary chromatography with capacitively coupled contactless conductivity detection and amperometric detection (MECC-C(4)D/AD). Electrophoretic runs were performed in a 35 mmol L(-1) SDS/60 mmol L(-1) H(3)BO(3)-Na(2)B(4)O(7) running buffer (pH 8.2) at a separation voltage of 16 kV, and five marker compounds and the major coexisting compound uric acid (UA) could be well separated within 23 min. Highly linear response was obtained for five marker compounds over three orders of magnitude with detection limits ranging from 6.6×10(-6) to 6.4×10(-8) g mL(-1) (S/N=3). The proposed method has been used to detect the marker compounds simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost. Urinary patterns in phenylketonuric babies were distinct and easily distinguished from those of healthy newborns. The proposed MECC-C(4)D/AD method could find clinical application in early noninvasive diagnosis of phenylketonuria (PKU), as significant differences could be found in the urinary content of five marker compounds among the phenylketonuric babies without or with dietotherapy and the healthy babies.

Topics: Chromatography, Micellar Electrokinetic Capillary; Electrochemical Techniques; Humans; Hydrogen-Ion Concentration; Infant, Newborn; Lactic Acid; Metabolome; Phenylacetates; Phenylketonurias; Phenylpyruvic Acids; Sodium Dodecyl Sulfate; Uric Acid

A novel method of CE coupled with dual electrochemical detection has been developed for the determination of pathological metabolites of phenylalanine in urine samples. Factors influencing the separation and detection were examined and optimized. Five aromatic acid metabolites and a major coexisting interfering compound uric acid could be well separated within 23 min at a separation voltage of 16 kV using a 35 mmol/L SDS/60 mmol/L H(3)BO(3)-Na(2)B(4)O(7) running buffer (pH 8.2). Highly linear response was obtained for these five biomarker compounds over three orders of magnitude with detection limits ranging from 6.6 to 0.064 μg/mL (S/N=3). The average recovery and RSD were within the range of 92.6-121.0 and 1.0-12.0%, respectively. The proposed method has been used to detect the unconjugated aromatic acids simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost, thus could find potential applications involving assays of biomarker compounds for the purpose of fast diagnose of some metabolic diseases including phenylketonuria.

Topics: Adult; Biomarkers; Electrodes; Electrophoresis, Capillary; Female; Humans; Hydrogen-Ion Concentration; Infant; Linear Models; Male; Phenylacetates; Phenylalanine; Phenylketonurias; Phenylpyruvic Acids; Pregnancy; Reproducibility of Results; Sensitivity and Specificity; Sodium Dodecyl Sulfate

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.

Topics: Adult; Animals; Cattle; Centrifugation, Density Gradient; Chelating Agents; Chromatography, DEAE-Cellulose; Chromatography, Gel; Copper; Electrophoresis, Polyacrylamide Gel; Fetus; Fluorine; Humans; Infant, Newborn; Iron; Kinetics; Liver; Molecular Weight; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Rats; Sodium Dodecyl Sulfate; Sulfides

Topics: Animals; Carbon Isotopes; Centrifugation; Humans; Kinetics; Liver; Male; Mixed Function Oxygenases; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Phosphoric Acids; Pteridines; Rats; Sodium Dodecyl Sulfate; Structure-Activity Relationship; Succinates; Tyrosine; Ultracentrifugation; Vitamin E