Distribution of some point mutations in the phenylalanine hydroxylase gene of phenylketonuria patients from the Moscow region.

Thirty-one unrelated phenylketonuria patients from the Moscow region were screened for mutations in the phenylalanine hydroxylase gene at the following codons: 408, 158, 261 and IVS-12. For detection of point mutations, polymerase chain reaction amplification was applied with allele-specific oligonucleotide hybridization. The following mutation frequencies were determined: codon 408-56.4%; codon 158-8.1%; codon 261-3.2%, and IVS-12-16%.

Identification of 4-methoxybenzoyl-N-glycine in urine by gas chromatography/mass spectrometry.

An unusual metabolite was detected in the urine of two children with neurological dysfunctions of unclear aetiology by using gas chromatography/mass spectrometry (GC/MS). On the basis of the analysis of its fragmentation pathways, synthesis of tentative compound and its GC/MS analysis it was stated that the unknown metabolite is 4-methoxybenzoyl-N-glycine.

Content of phenylalanine, tyrosine and their metabolites in CSF in phenylketonuria.

By using ion-exchange chromatography and gas chromatography coupled with mass spectrometry, the content of phenylalanine, tyrosine and their metabolites typical of phenylketonuria (PKU) was determined in the cerebrospinal fluid (CSF) of 8 untreated children with classical PKU and 9 controls. At the same time, plasma and urine were analysed. In PKU the content of phenylalanine is increased on average 23 times in plasma and CSF. The content of phenylalanine and tyrosine in CSF is about 4 times less as compared with plasma. The phenylalanine-to-tyrosine ratio is approximately the same for these fluids both in control and in PKU. This indicates that the transport of phenylalanine and tyrosine through the blood-brain barrier is not disturbed in PKU. Phenylpyruvate and 4-hydroxyphenylpyruvate are either not detected or present in very low concentrations in the CSF of children with PKU; their derivatives, phenyllactate and 4-hydroxyphenyllactate, are present in relatively higher concentrations. This indicates increased metabolic conversion in brain tissues.

[The recording of human fibroblast metabolism in vitro by the chromatographic-mass spectrometric determination of the metabolites excreted]. / Registratsiia metabolizma fibroblastov cheloveka in vitro putem khromato-mass-spektrometricheskogo opredeleniia ékskretiruemykh imi metabolitov.

Metabolites excreted into culture medium by human skin fibroblasts have been studied by high resolution gas chromatography and mass spectrometry. Parameters for 29 metabolites have been obtained and 11 of them have been identified. Excreted metabolites reflect activity of certain metabolic processes in fibroblasts. Comparison of chromatographic and mass spectrometric parameters of cellular metabolites with the metabolites excreted with urine revealed that most metabolites excreted from fibroblasts differ from urine metabolites. The possibility for secondary transformation of cell metabolites in organism and specificity of metabolism in different tissues has been discussed.

Discovery of methoxyacetylcarbamide in the urine of normal adults and phenylketonuric children.

In order to study metabolic distinctions in phenylketonuria, urinary metabolites in the form of trimethylsilyl derivatives have been characterized by high resolution gas chromatography and mass spectrometry. A previously unknown metabolite has been found in the urine of some untreated phenylketonuric infants between 2 and 5 yr of age. The metabolite was absent in healthy children of the same age. The metabolite appeared to be present in the urine of apparently healthy adults (25-32 yr old). The metabolite was identified as methoxyacetylcarbamide on the basis of mass fragmentation analysis and compared with synthetic methoxyacetylcarbamide. Their retention times and mass spectra coincided.

Determination of human fibroblasts metabolism in vitro by gas chromatography-mass spectrometry of cell-excreted metabolites.

In order to develop an approach to the study of cell metabolism in vitro, we undertook the determination of metabolites excreted by human skin diploid fibroblasts into culture medium using high-resolution gas chromatography in combination with mass spectrometry. Chromatographic and mass spectrometric characteristics of 29 metabolites have been obtained, and 11 of the metabolites have been identified. The excreted metabolites reflect the activity of certain metabolic processes in fibroblasts in vitro. A comparison of chromatographic and mass spectrometric characteristics of the cell metabolites and of those excreted from the body in urine showed most of the metabolites excreted by fibroblasts to be different from the urine metabolites. The possibility of secondary conversion of cell metabolites in the organism and the specificity of metabolism in cells of different tissues are discussed.

Excretion of phenylpyruvic, 4-hydroxyphenylpyruvic and indolyl-3-acetic acids by the skin fibroblasts from a phenylketonuric child.

High-resolution gas chromatography and mass spectrometry have been used to analyse the changing chemical composition of the culture medium upon the growth of skin fibroblasts from a healthy individual and from a classical phenylketonuric (PKU) patient. The PKU fibroblasts, unlike normal ones, were found to excrete into the culture medium phenylpyruvate, 4-hydroxyphenylpyruvate, indolyl-3-acetate and an unidentified metabolite containing the phenyl group. This testifies to the disturbed metabolism of phenylalanine, tyrosine and tryptophan in the PKU fibroblasts. This seems to be the reason for their reduced viability as compared with normal fibroblasts.