Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS).

The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min. To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1). In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.

Sensitive nonradioactive dot blot/ribonuclease protection assay for quantitative determination of mRNA.

We have developed a simple and sensitive method for the rapid quantitation of mRNA from cell cultures and small tissue samples. The method combines the high sensitivity and specificity of the ribonuclease protection assay with simple handling and rapid execution of dot blotting. The use of digoxygenin-labeled cRNA probes eliminates all problems associated with radioisotopes commonly used in the ribonuclease protection assay. The RNA preparation is dotted directly onto nylon membranes, and after hybridization the filters are treated with ribonuclease A, which removes the nonhybridized single-stranded RNA. The mRNA-hybrid is then visualized by the chemiluminescence technique using labeled anti-digoxigenin antibody, and the signal intensity is quantitated. Comparison with the Northern blotting ribonuclease protection assay revealed that this dot blot technique is almost ten times more sensitive and that its signals are linear over a wide range of RNA concentrations (0.01-10 micrograms/microL/dot). This method seems particularly valuable for simultaneous processing of large numbers of samples containing a wide range of RNA concentrations.

Measurement of vitellogenin gene expression by RT-PCR as a tool to identify endocrine disruption in Japanese medaka (Oryzias latipes).

In order to monitor vitellogenin gene expression in the Japanese medaka (Oryzias latipes), a reverse transcription-polymerase chain reaction (RT-PCR) system was developed. To date cDNA for medaka vitellogenin has not been published; therefore, initially a sequence fragment had to be obtained and compared with other known vertebrate vitellogenins. For this, a 1.2 kb cDNA of medaka vitellogenin (M-Vg1.2) was amplified by RT-PCR and cloned into a pCRR H-TOPO bacterial vector. On Northern blot analysis, the antisense cRNA of M-Vg1.2 stained a 5.5 kb gene product found exclusively in female fish, but not in males. Additionally, the 5'-end of medaka vitellogenin cDNA was amplified by 5'-RACE-PCR. The analysed nucleotide sequence of 1.6 kb shared significant similarities with vitellogenins known from other fish species: approximately 72% similarity with mummichog (Fundulus heteroclitus) vitellogenin I and approximately 62% with fathead minnow (Pimephales promelas) vitellogenin. To develop a semiquantitative RT-PCR for the measurement of vitellogenin gene expression, primers specific to a 500 bp sequence of the vitellogenin cDNA (M-Vg0.5) were constructed using the gene product of elongation factor 1 alpha as internal standard. Induction of vitellogenin gene expression was measured in male medaka exposed to 0, 2, 20 and 50 micrograms l-1 nonylphenol and 0, 2.5, 25 and 100 ng l-1 17 alpha-ethinyloestradiol for 7 days. The LOECs for vitellogenin induction in male medaka were 20 micrograms l-1 and 25 ng l-1 for nonylphenol and 17 alpha-ethinyloestradiol, respectively.

Localization of the heme-binding protein in the cytoplasm and of a heme-binding protein-like immunoreactive protein in the nucleus of rat liver parenchymal cells: immunocytochemical evidence of the subcellular distribution corroborated by radioimmunoassay and immunoblotting.

The abundant heme-binding protein of the liver, probably identical with Z-protein or liver fatty acid-binding protein, has an apparent molecular weight of 14,000 Da and is presumably involved in the intracellular transport of a variety of compounds. The cellular and subcellular distribution of HBP in the liver was studied in adult male and female rats by postembedding immunocytochemistry using the protein A-gold technique. By light microscopic examination heme-binding protein is present exclusively in parenchymal cells and not found in the sinusoidal lining cells or other cells in portal tracts. Immunoreactivity for heme-binding protein is uniformly strong throughout the liver lobule in female rats but is markedly reduced in the pericentral region in male animals. By immunoelectron microscopy heme-binding protein immunoreactivity is localized in cytoplasm and nuclear matrix. The mitochondria and peroxisomes and the secretory apparatus are free of the label. In nuclei, gold labeling is confined to the interchromatin region (euchromatin) and nucleoli; condensed chromatin (heterochromatin) and nucleolus-associated chromatin are negative. The subcellular localization was substantiated by radioimmunoassay and immunoblotting of nuclear and cytosolic fractions. Immunoblotting shows that the heme-binding protein-like immunoreactive protein in the nucleus has a slightly larger molecular weight than that in the cytoplasm.