Biomedical subjects
Publications and source records attributed to K Hyland.
This review briefly describes the biochemistry of pterins, their involvement in pathological processes and the use of pterin measurement in diagnosis and monitoring of disease. Chromatographic and other methods of pterin analysis are detailed with particular emphasis being placed on the need for correct sample collection and handling.
We previously described demyelination in the brain and subacute combined degeneration of the spinal cord in a patient with 5,10-methylenetetrahydrofolate reductase deficiency. To assess the role of methionine, S-adenosylmethionine, folate, and neurotransmitter amine metabolism in the demyelination process, we measured these metabolites in CSF from this patient; the findings are compared with those obtained from three patients in whom neurologic deterioration had been halted by the administration of betaine. Folate concentrations were low, and amine and biopterin metabolism were abnormal in all patients. Methionine and S-adenosylmethionine concentrations were undetectable in the first patient. In those receiving betaine, methionine concentrations were proportional to the dose administered and S-adenosylmethionine concentrations were near normal. The results provide the first evidence for an association between defective S-adenosylmethionine metabolism and demyelination in humans.
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Measurement of tetrahydrobiopterin in cerebrospinal fluid requires careful handling of samples during storage and analysis. Addition of dithioerythritol and deoxygenation of the mobile phase with helium prevents breakdown of tetrahydrobiopterin during chromatography. Tetrahydrobiopterin in cerebrospinal fluid is unstable at room temperature, 100% being lost within 3.5 h, this breakdown does not generate equivalent quantities of dihydrobiopterin and biopterin. Addition of dithioerythritol and diethylenetriaminepenta-acetic acid to cerebrospinal fluid prevents breakdown of tetrahydrobiopterin for 6 mth at -70 degrees C and for up to 5 h at 4 degrees C. At room temperature less than 5% of tetrahydrobiopterin was lost after 2 h.
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We describe an isocratic, reversed-phase high-performance liquid chromatographic method for the simultaneous measurement of fully oxidised, dihydro- and tetrahydropterins in cerebrospinal fluid. Tetrahydrobiopterin is detected electrochemically using an ESA Coulochem detector in the redox mode. Dihydropterins are detected by fluorescence following post-column electrochemical oxidation, and fully oxidised pterins by their natural fluorescence. Apart from addition of antioxidants, no sample preparation is required. Comparison is made with methods requiring chemical oxidation for detection of tetrahydrobiopterin. Some results on children with neurological disease are presented.
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A 3-month-old girl presented with anorexia, failure to thrive and drowsiness. She was mildly icteric with hepatomegaly and peripheral oedema. Disordered liver function tests were associated with the biopsy appearances of a giant cell hepatitis and with a Fanconi syndrome. At the age of 16 weeks she collapsed with profound hypoglycaemia. Fasting also provoked hypoglycaemia with lactic acidaemia. She became increasingly irritable and hypotonic and, although initially liver and renal function improved, she deteriorated and died of hepatocellular failure and septicaemia. A post-mortem revealed massive fatty degeneration of the liver. The activity of phosphoenolpyruvate carboxykinase in her cultured skin fibroblasts was 16% of controls. Her brother died at the age of 4 weeks of sudden infant death syndrome.
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To test the hypothesis that some of the neurologic sequelae of treatment for acute lymphoblastic leukemia (ALL) might be related to abnormalities in biopterin metabolism associated with methotrexate (MTX) therapy, total biopterin levels in cerebrospinal fluid (CSF) and plasma, and homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) were measured in a cross-sectional study of 80 children with ALL. For comparison, biopterins were also measured in a group of children of similar age undergoing investigation for neurologic disease. In children with ALL studied before therapy, no significant difference was found between the means of plasma biopterin or CSF biopterin concentrations and the means in the control group. In children receiving MTX, plasma biopterin values were higher in the group given maintenance therapy than in children observed before treatment. CSF levels were significantly increased only in those patients who had completed 2 years of maintenance therapy. CSF concentrations of HVA and 5HIAA in patients with ALL who had received no treatment (median values 52 and 18 ng/ml, respectively) showed a wide scatter and were inversely related to age. In patients receiving MTX, concentrations of these metabolites were higher than in the untreated group, again reaching a peak in patients just completing 2 years of treatment (median HVA 110 ng/ml, 5HIAA 34 ng/ml). These results provide no support for the idea that neurotransmitter amine deficiency occurs in children with ALL receiving MTX, and indicate, rather, that amine and biopterin synthesis increases in such patients.
Plasma total biopterin concentration was measured by bioassay in 59 infants with hyperphenylalaninaemia and in 50 children with developmental regression and or movement disorder with normal plasma phenylalanine concentrations. In infants with raised phenylalanine concentrations plasma biopterin concentrations were significantly raised in proportion to the phenylalanine values. Five patients had plasma biopterin concentrations at the extremes of the range, and of these two had defective biopterin metabolism. One with low plasma biopterin concentration apparently had a partial defect of biopterin synthesis but died before investigations were complete. One with high plasma biopterin concentration, even when phenylalanine concentrations had fallen to the normal range, had dihydropteridine reductase deficiency. In this patient concentrations of homovanillic acid and 5-hydroxyindolacetic acid in the cerebrospinal fluid (CSF) were severely reduced. In children without hyperphenylalaninaemia plasma biopterin concentrations were normal. Twenty two patients were subjected to lumbar puncture, of whom six with developmental regression without movement disorder had normal CSF biopterin concentrations, and 11 with movement disorder other than torsion dystonia had significantly lower CSF biopterin concentrations. Five patients with torsion dystonia had normal biopterin concentrations.
A 2-year-old girl with 5,10-methylenetetrahydrofolate reductase deficiency developed subacute combined degeneration of the cord and a leuco-encephalopathy which was confirmed at necropsy. Total folate concentrations in serum, red cells and CSF were markedly reduced whereas vitamin B12 concentrations were normal. In addition the patient had Parkinsonism and reduced concentrations of homovanillic acid, 5-hydroxyindoleacetic acid and total biopterins in cerebrospinal fluid. Folic acid administration was accompanied by fits and acute deterioration in the movement disorder. At necropsy the basal ganglia showed no detectable abnormality.
Patients with phenylalanine hydroxylase deficiency show increased concentrations of biopterins and neopterins, and reduced concentrations of serotonin and catecholamines, when phenylalanine concentrations are raised. The pterin rise reflects increased synthesis of dihydroneopterin and tetrahydrobiopterin, and the amine fall a reduction in amine synthesis due to inhibition by phenylalanine of tyrosine and tryptophan transport into neurones. The pterin and amine changes appear to be independent of each other and are present in the central nervous system as well as the periphery; they disappear when phenylalanine concentrations are reduced to normal. Patients with arginase deficiency show a similar amine disturbance but have normal pterin levels. The amine changes probably contribute neurological symptoms but pterin disturbance is not known to affect brain function. Patients with defective biopterin metabolism exhibit severely impaired amine synthesis due to tetrahydrobiopterin deficiency. Pterin concentrations vary with the site of the defect. Symptoms include profound hypokinesis and other features of basal ganglia disease. Neither symptoms nor amine changes are relieved by controlling phenylalanine concentrations. Patients with dihydropteridine reductase (DHPR) deficiency accumulate dihydrobiopterins and develop secondary folate deficiency which resembles that occurring in patients with defective 5,10-methylene tetrahydrofolate reductase activity. The latter disorder is also associated with Parkinsonism and defective amine and pterin turnover in the central nervous system, and a demyelinating illness occurs in both disorders. In DHPR deficiency cerebral calcification may develop in a similar distribution to that seen in congenital folate malabsorption and methotrexate toxicity. Symptoms are ameliorated by therapy with 5-formyltetrahydrofolate but exacerbated by folic acid.
A method is described for the separation and detection of tetrahydro, dihydro and fully oxidised pterins in a single chromatographic run using ion-pair reversed-phase high-performance liquid chromatography. Tetrahydropterins are detected by electrochemical oxidation, dihydropterins by fluorescence following post-column electrochemical oxidation and the fully oxidised pterins by their natural fluorescence. The post-column electrochemical conversion of the non-fluorescent dihydropterins to fluorescent compounds is proportional to the amount injected over three orders of magnitude. Because of the relative selectivity of the fluorescence detection and the low potential required to oxidise the tetrahydropterins, all the oxidation species of the pterins may be measured in biological samples with minimal sample clean-up.
In most patients with deficiency of tetrahydrobiopterin (BH4) continuous administration of BH4 or of a synthetic analogue such as 6-methyltetrahydropterin (6-MPH4) lowers plasma phenylalanine concentrations to the therapeutic range. The effective dose of BH4 varies from 1 to 2 mg kg-1 daily in patients with defective biopterin synthesis, to 5 mg kg-1 or more in patients with dihydropteridine reductase (DHPR) deficiency. The cost of 2 mg kg-1 day-1 of BH4 is comparable to the cost of a low phenylalanine diet. Higher doses of pterins given orally (20 mg kg-1) raise the levels of tetrahydropterin in cerebrospinal fluid (CSF) to normal in patients with defective biopterin synthesis in whom initial concentration of biopterin species are low. In some, but not all, such patients pterin therapy also raises CSF amine metabolite concentrations and ameliorates symptoms. High dose therapy does not appear to be effective in raising CSF pterin levels in patients with DHPR deficiency who already accumulate dihydrobiopterin (BH2) in CSF. Central folate deficiency is an additional cause of neurological deterioration in patients with DHPR deficiency who require supplementation with folate as folinic acid. It is suggested that the accumulation of BH2 in such patients competitively interferes with folate metabolism.
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