Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal beta-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2-48 nm, 3-hydroxypristanic acid: 0.02-0.81 nm, 3-ketopristanic acid: 0.07-1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.-Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometr

Cytogenetic characterization of telomeres in the holocentric chromosomes of the lepidopteran Mamestra brassicae

Telomeres of the Mamestra brassica holocentric chromosomes were studied by Southern blotting, in-situ hybridization and Bal31 assay evidencing the presence of the telomeric (TTAGG)(n) repeat. Successively, molecular analysis of telomeres showed that TRAS1 transposable elements were present at the subtelomeric regions of autosomes but not in the NOR-bearing telomeres of the Z and W sex chromosomes. TRAS1 appeared to be transcriptionally active and non-methylated, as evaluated by RT-PCR and digestion with MspI and HpaII. Finally, dot-blotting experiments showed that the 2.8 +/- 0.5% of the M. brassicae genome consists of TRAS1

A phase ib clinical trial of metformin and chloroquine in patients with IDH1-mutated solid tumors

Simple SummaryMutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in high-grade chondrosarcoma, high-grade glioma and intrahepatic cholangiocarcinoma. Due to the lack of effective treatment options, these aggressive types of cancer have a dismal outcome. The metabolism of IDH1-mutated cancer cells is reprogrammed in order to produce the oncometabolite D-2-hydroxyglutarate (D-2HG). In this clinical trial, we used the oral antidiabetic drug metformin and the oral antimalarial drug chloroquine to disrupt the vulnerable metabolism of IDH1-mutated solid tumors. We found that the combination regimen of metformin and chloroquine is well tolerated, but the combination did not induce a clinical response in this patient population. Secondly, we confirmed the clinical usefulness of D/L-2HG ratios in serum as a biomarker and the ddPCR-facilitated detection of an IDH1 mutation in circulating DNA from peripheral blood.Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in 60% of chondrosarcoma, 80% of WHO grade II-IV glioma and 20% of intrahepatic cholangiocarcinoma. These solid IDH1-mutated tumors produce the oncometabolite D-2-hydroxyglutarate (D-2HG) and are more vulnerable to disruption of their metabolism. Methods: Patients with IDH1-mutated chondrosarcoma, glioma and intrahepatic cholangiocarcinoma received oral combinational treatment with the antidiabetic drug metformin and the antimalarial drug chloroquine. The primary objective was to determine the occurrence of dose-limiting toxicities (DLTs) and the maximum tolerated dose (MTD). Radiological and biochemical tumor responses to metformin and chloroquine were investigated using CT/MRI scans and magnetic resonance spectroscopy (MRS) measurements of D-2HG levels in serum. Results: Seventeen patients received study treatment for a median duration of 43 days (range: 7-74 days). Of twelve evaluable patients, 10 patients discontinued study medication because of progressive disease and two patients due to toxicity. None of the patients experienced a DLT. The MTD was determined to be 1500 mg of metformin two times a day and 200 mg of chloroquine once a day. A serum D/L-2HG ratio of >= 4.5 predicted the presence of an IDH1 mutation with a sensitivity of 90% and a specificity of 100%. By utilization of digital droplet PCR on plasma samples, we were able to detect tumor-specific IDH1 hotspot mutations in circulating tumor DNA (ctDNA) in investigated patients. Conclusion: Treatment of advanced IDH1-mutated solid tumors with metformin and chloroquine was well tolerated but did not induce a clinical response in this phase Ib clinical trial.Molecular tumour pathology - and tumour geneticsMTG

Automated determination of pyrethroid insecticides in surface water by column liquid chromatography with diode array UV detection, using on-line micelle-mediated sample preparation.

A liquid chromatographic method using gradient elution and diode array UV detection for the trace level determination of seven synthetic pyrethroids is described. The procedure involves automated on-line preconcentration on precolumns containing octadecylbonded silica using a Prospekt. Although the analytes display a "mixed" hydrophobic/hydrophilic behaviour, both breakthrough on the precolumn and adsorption to inner walls and surfaces can be prevented by adding Brij-35, a neutral surfactant, to the aqueous sample. The procedure has been tested for the analysis of surface water; detection limits typically are at the sub-μg/l level and repeatability is excellent. The automated system is robust and there are no maintenance problems. © 1994 Springer-Verlag

Pyridosineafhankelijke epilepsie.

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Pyridoxine-dependent seizures in Dutch patients: diagnosis by elevated urinary alpha-aminoadipic semialdehyde levels.

Contains fulltext : 52938.pdf (publisher's version ) (Closed access)BACKGROUND: Pyridoxine-dependent seizures (PDS) is a rare, autosomal recessively inherited disorder. Recently alpha-aminoadipic semialdehyde (alpha-AASA) dehydrogenase deficiency was identified as a major cause of PDS, which causes accumulation of both alpha-AASA and pipecolic acid (PA) in body fluids. METHODS: We studied urinary and plasma alpha-AASA and PA levels in 12 Dutch clinically diagnosed patients with PDS. RESULTS: Alpha-AASA was elevated in both urine and plasma in 10 patients. In these patients plasma PA levels were also elevated but urinary PA levels were normal. DISCUSSION: In all patients with clinically definite PDS, and in most patients with probable or possible PDS, the clinical diagnosis of PDS could be confirmed at the metabolite level. Non-invasive urinary screening for alpha-AASA accumulation provides a reliable tool to diagnose PDS and can save these patients from the classical and potentially dangerous pyridoxine withdrawal test to prove PDS