An online solid-phase extraction-liquid chromatography-tandem mass spectrometry method to study the presence of thyronamines in plasma and tissue and their putative conversion from 13C6-thyroxine.

Thyronamines are exciting new players at the crossroads of thyroidology and metabolism. Here, we report the development of a method to measure 3-iodothyronamine (T(1)AM) and thyronamine (T(0)AM) in plasma and tissue samples. The detection limit of the method was 0.25 nmol/l in plasma and 0.30 pmol/g in tissue both for T(1)AM and for T(0)AM. Using this method, we were able to demonstrate T(1)AM and T(0)AM in plasma and liver from rats treated with synthetic thyronamines. Although we demonstrated the in vivo conversion of (13)C(6)-thyroxine ((13)C(6)-T(4)) to (13)C(6)-3,5,3'-triiodothyronine, we did not detect (13)C(6)-T(1)AM in plasma or brain samples of rats treated with (13)C(6)-T(4). Surprisingly, our method did not detect any endogenous T(1)AM or T(0)AM in plasma from vehicle-treated rats, nor in human plasma or thyroid tissue. Although we are cautious to draw general conclusions from these negative findings and in spite of the fact that insufficient sensitivity of the method related to extractability and stability of T(0)AM cannot be completely excluded at this point, our findings raise questions on the biosynthetic pathways and concentrations of endogenous T(1)AM and T(0)AM.

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus.

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18,174 Da. The molecular mass of the purified protein was estimated to be 18,176. +/ 0.80 Da by electrospray mass spectrometry. The bfr was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conversed in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Iron metabolism in Rhodobacter capsulatus. Characterisation of bacterioferritin and formation of non-haem iron particles in intact cells.

The water-soluble cytochrome b557 from the photosynthetic bacterium Rhodobacter capsulatus was purified and shown to have the properties of the iron-storage protein bacterioferritin. The molecular mass of R. capsulatus bacterioferritin is 428 kDa and it is composed of a single type of 18-kDa subunit. The N-terminal amino acid sequence of the bacterioferritin subunit shows 70% identity to the sequence of bacterioferritin subunits from Escherichia coli, Nitrobacter winogradskyi, Azotobacter vinelandii and Synechocystis PCC 6803. The absorbance spectrum of reduced bacterioferritin shows absorbance maxima at 557 nm (alpha band), 526 nm (beta band) and 417 nm (Soret band) from the six haem groups/molecule. Antibody assays reveal that bacterioferritin is located in the cytoplasm of R. capsulatus, and its levels stay relatively constant during batch growth under aerobic conditions when the iron concentration in the medium is kept constant. Iron deficiency leads to a decrease in bacterioferritin and iron overload leads to an increase. Bacterioferritin from R. capsulatus has an amorphous iron-oxide core with a high phosphate content (900-1000 Fe atoms and approximately 600 phosphates/bacterioferritin molecule). Mössbauer spectroscopy indicates that in both aerobically and anaerobically (phototrophically) grown cells bacterioferritin with an Fe3+ core is formed, suggesting that iron-core formation in vivo may not always require molecular oxygen.

Comparison of cytosolic products formed in rat liver in response to parenteral and dietary iron loading.

Two different methods were used to create a situation of iron (Fe) overload in rats. One group of rats received Fe dextran, and another group of rats received a carbonyl Fe-enriched diet. The ferritins present in the liver cytosol of these rats were isolated and compared. From each group, two cytosolic products were isolated with the use of ultracentrifugation: a cytosolic ferritin fraction (CF) and a (slower sedimenting) light ferritin fraction (CLF). There were no differences with respect to the protein coat (subunit composition and amino acid analysis). Analysis of the Fe core revealed that the two CF fractions were similar, whereas the two CLF fractions differed with respect to their Fe content and to the packing of their cores. The carbonyl CLF product contained less Fe atoms/molecule, which, moreover, seemed to be packed in a less compact way.

In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.

Quantitative energy-filtered image analysis in cytochemistry. II. Morphometric analysis of element-distribution images.

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per element relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.

Analysis of iron-containing compounds in different compartments of the rat liver after iron loading.

The livers of iron-loaded rats were fractionated and a cytosolic fraction, a lysosomal fraction, a siderosomal fraction and haemosiderin were obtained. All iron-containing compounds from these fractions were isolated and their morphology, Fe/P ratios, iron core diameter and peptide content were compared. The cytosolic fraction contained ferritin (CF) and a slower sedimenting, light ferritin (CLF). The lysosomal fraction also contained ferritin (LF) and a slower sedimenting light ferritin (LLF). The siderosomal fraction contained ferritin (SF), a faster sedimenting non-ferritin iron compound (SIC) and haemosiderin (HS). SIC and HS did not resemble ferritin as much as the other products did, but were found to be water-insoluble aggregates. The Fe/P ratios of CF and CLF were lower than the Fe/P ratios of LF and LLF and these in turn had lower Fe/P ratios than SF, SIC and HS. The iron core diameter of the cytosolic ferritin was increased after lysosomal uptake. The iron core diameters of the siderosomal products were smaller. CLF, CF, LF, LLF and SF contained one kind of subunit of approximately 20.5 kDa. SIC and HS contained other peptides in addition to the 20.5-kDa subunit. The results indicate that storage of ferritin molecules is not limited to the cytosolic compartment, but is also the case in the lysosomes. Extensive degradation of the ferritin molecule seems to be confined to the siderosomes.

Lysosomal and cytosolic ferritins. A biochemical and electron-spectroscopic study.

Cytosolic and lysosomal ferritin and haemosiderin were isolated from rat livers which had been iron-loaded by four intraperitoneal injections of iron-dextran. The cytosolic and lysosomal ferritins, prepared in a phosphate-free medium, were subjected to gel-filtration chromatography on Sepharose 6B, yielding four fractions: a cytosolic monomeric (CMF) and void-volume ferritin fraction (CVVF), and a lysosomal monomeric (LMF) and void-volume ferritin fraction (LVVF). Of each fraction the following aspects were examined: (a) immunoreactivity against specific antiserum; (b) the Fe/P mass ratio and the effect of dialysis on this ratio using electron probe micro-analysis (EPMA); (c) morphology and Fe-specific imaging using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). For haemosiderin one aspect, the Fe/P ratio, was determined before and after extensive purification. The following results were obtained (a) All ferritin fractions reacted with anti- (rat liver ferritin). (b) The Fe/P ratios as determined in CMF in an haemosiderin were not affected by dialysis or extensive purification, respectively. The Fe/P ratio in CVVF was affected by dialysis. In the lysosomal fractions, only a trace of phosphorus (LVVF) or no phosphorus (LMF) was detected. (c) Morphologically, CMF and CVVF were found to be rather homogeneous; the iron core diameters of both fractions were in the known size range.(ABSTRACT TRUNCATED AT 250 WORDS)