High-throughput non-heme iron assay for animal tissues

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.     

Influence of ashing techniques on the analysis of trace elements in animal tissue

A multitude of methods exists at present for the solubilization of biological tissues for atomic absorption analysis. We have examined several common methods of wet ashing using NBS bovine liver in order to determine which acids, acid combinations, or bases should be used as digesting agents for accurate and precise measurement of iron, copper, zinc, and manganese. Nitric acid proved to be the most effective wet ashing agent. With nitric acid, mean concentrations for iron, copper, and zinc differed from NBS certified values by less than 1.5% while those for manganese differed by 4%.     

Asbestos-stimulated changes in nitric oxide and iron metabolism in rats

Under intratracheal asbestos fibers installation it has been investigated NO synthesis in the lung and liver tissues of Wistar rats by EPR method. Asbestos A6-45, sifted through the sieve with size 0.1 mm, has been administrated in a dose of 5 mg/kg. To evaluate the NO synthesis EPR and NO-trap methods have been used. The amplitude of EPR signal "trap-NO" in the lung samples was 12, 16 and 14 times greater than in controls on the 3th, 6th and 10th days after asbestos installation and was corresponding to NO rate of about 2 mkmol/(g x h). In the liver samples of asbestos-stimulated animals the NO level contained in the non-heme iron nitrosyl complexes was about 2 mkmol/g. Thus, the asbestos fibers stimulate NO synthesis not only in the lung tissue, but also in other organs. The obtained data shows that under NO hyperproduction certain changes in iron metabolism take place, such as: the decrease of transferrin iron and the accumulation of ferric iron not bound with transferrin. The accumulation of ferric iron not shielded by proteins is one of the oxidative stress triggers.     

A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues

In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one flu-orometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 µmole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 µmoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSC determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

Gulonolactone oxidase is missing in teleost fish. The direct spectrophotometric assay

Data in the literature imply that some fish species evolved with the capacity to synthesize ascorbic acid. Gulonolactone oxidase activity has been reported in kidney and/or liver tissues. However, it is shown here that this microsomal enzyme activity is missing in common carp hepatopancreas and kidney, whereas high activity was confirmed in pigeon kidney, rat liver, bovine liver and amphibian (Xenopus) kidney tissues. A new assay using either the whole tissue homogenate or microsomes solubilized by sodium deoxycholate was developed to directly measure the formation of ascorbic acid spectrophotometrically. Identical values were found using this assay as well as the assay in which formed ascorbate was determined by the dinitrophenyl hydrazine (DNPH) method. In some experiments, these results were confirmed by polarographically measured oxygen consumption.     

Indices of oxidative stress. 2. Lipid peroxides

Two methods of the determination of lipid peroxidation products have been compared which are based on Fe(II) oxidation by them at acid pH values in the presence of xylenol orange which binds Fe(III) have been compared. The first method uses cumene hydropeoxide as an internal standard. In the second one, lipid peroxides are previously reduced by triphenylphosphine and these substances content is measured as a difference of the production of complexes with xylenol orange and iron ions in the control (with reduction) and experimental sample (without reduction). The optimization of measurement conditions is described. The levels of lipid peroxides in goldfish tissues assayed simultaneously by two methods were similar. The method with cumene hydroperoxide needs less amounts of biological material; moreover, there is no necessity in a calibration curve. Effects of hyperoxia on lipid peroxide levels in goldfish tissues were studied with the cumene method. Within the first hours of hyperoxia this index increased 13-times in the liver and 2-times in the brain and muscle. The further exposure rebounded this parameter to the initial level. Levels of lipid peroxides positively correlated with levels of end products of lipid peroxidation (thiobarbiturate acid reactive substances) in the goldfish tissues. The method of quantification of lipid peroxides with cumene is recommended for wide using in biological investigations.     

Measurement of loosely-bound iron in brain regions using redox cycling and salicylate.

A sensitive iron assay was developed for measuring non-heme and loosely bound iron in regions of rat brain. The method is based on the salicylate trapping of hydroxyl radicals generated from ascorbate-driven redox cycling of Fe3+-EDTA. This assay has high sensitivity (about 20 nM) because of amplification obtained with redox-cycling and fluorescent detection of the salicylate hydroxylation product, 2,5-dihydroxybenzoate. The assay detects iron as Fe2+ and Fe3+ combined. Values of non-heme and loosely bound iron are given for three areas of cortex, caudate, hippocampus, thalamus and brainstem of the rat brain.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

Increased intestinal iron absorption in rats with normal hepatic iron stores. Kinetic aspects of the adaptative response to parenteral iron repletion in dietary iron deficiency

Male Sprague-Dawley rats were fed an iron-deficient diet for 8 days. After this period, iron stores were repleted in three groups of animals by intravenous administration of iron dextran. In a second set of experiments, iron was administered in the same dose as Fe nitrilotriacetic acid complex. 12 h, 24 h and 48 h thereafter, the intestinal iron transfer in vitro and in vivo as well as the non-heme iron and ferritin content were determined in both the liver and the jejunal mucosa. In iron deficiency, intestinal iron transfer is increased to 230% of untreated controls, while non-heme iron and ferritin decreased to 20% and 10% in the liver and to 55% and 25% in the mucosa, respectively. 12 h and 24 h after parenteral administration of 0.1 mmol Fe/kg body weight iron transfer was as high as in iron deficiency, while liver iron stores were not significantly different from the untreated controls. In this situation, the close link between decreases in body iron stores and increases in iron transfer was temporarily dissociated. This can be related to the time lag between the incorporation of parenterally applied iron in the liver and in the jejunal mucosa. The data provide evidence for the hypothesis that the hepatic iron stores have no means of neural or hormonal communication with the small intestine in order to adapt iron transfer to their state of repletion on short notice. Intestinal iron transfer returned to control levels after 48 h.     

A sensitive enzymatic procedure for the quantitative determination of S-adenosylhomocysteine in tissues

A simple, sensitive, and specific method for analysis of S-adenosyl-l-homocysteine (SAH) in tissue is described. The assay is based on the measurement of the inhibitory activity of SAH on catechol-O-methyltransferase (COMT). As little as 1 mg of fresh liver tissue may be used. The precedure involves extraction of tissues, thin-layer chromatographic separation, followed by measurement of COMT. The concentration of SAH in rat liver assessed by this method is 71.2 ± 3.8 nmol/g of wet tissue and is principally concentrated in the particulate fractions.     

Iron sources forming nitrosyl complexes in animal tissues

Treatment of perfused mouse liver with nitric oxide does not change the intensities of ESR signals of iron-sulfur proteins characteristic of this tissue. Proceeding from this evidence and also from the ratio between the iron content in these proteins and dinitrosyl iron complexes (complexes 2.03) formed in the liver when it contacts with NO, it is concluded that iron-sulfur proteins are not involved in the formation of complexes 2.03. It seems that only the loosely bound form of non-heme iron-free iron is involved in this process.     

Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.     

Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state

A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed 'non-heme non-FeS iron', is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K. The 'non-heme non-FeS iron', which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 +/- 0.3 nmol . mg-1 protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 +/- 0.2 nmol . mg-1 protein) is localized to the 'outer' compartment and a major fraction (i.e. 1.1 +/- 0.1 nmol . mg-1 protein) is localized to the 'inner' compartment and is equally distributed between the inner membrane and the matrix. The experiments described in this study also indicate that approximately half of the 'non-heme non-FeS iron' of the 'inner' pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Hemosiderin. an EPR study of water-insoluble iron in human and rat liver.

EPR spectra of the water-insoluble iron fraction, hemosiderin of human and rat liver are described. The homogenate of freshly prepared perfused rat liver shows a non-heme iron signal at g=4.3 and a high-spin heme-iron signal around g=6, whereas the washed and sonicated sample of the insoluble iron fraction shows solely a non-heme iron signal at g=4.3. This indicates that hemosiderin from rat liver does not contain heme iron. Human-liver preparations from post mortem obtained material show in the homogenates as well as in the washed and sonicated samples an intense high-spin heme iron signal at g=6.0 and a non-heme iron signal at g=4.3. A comparative experiment, carried out with "aged" rat liver preparations, reveals the same spectra as with the human preparations. It is concluded that that the heme present in the insoluble iron fraction is caused by degradation of hemoglobin in the obduction material, and that heme is not a constituent of the insoluble depot iron.     

A micromethod for the enzymatic estimation of the degree of glycogen ramification

A comparison of methods for the evaluation of glycogen content in liver tissue of rats has been carried out by determining the recoveries in the differential ethanol precipitation of glycogen from alkaline tissue digests as well as the actual quantitative equivalence between glycogen content and actual glucose measured. Hydrolytic/enzymatic methods gave lower results than non-specific chemical methods such as anthrone. These lower values, combined with the losses in the purification process resulted in much lower glycogen estimations than the actual estimated tissue content. A method has been devised for the measurement of glycogen ramification in small liver tissue samples, using neutral periodate oxidation of the molecule, followed by determination of the formic acid evolved from the branch ends with formic acid dehydrogenase. The method gave results very similar to the classical methods in which the acid formed is measured titrimetrically. Rat liver tissue contained a mean 323 +/- 69 mmol of glucose equivalents of glycogen per gram of tissue; this glycogen had a mean chain length of 11.4 +/- 0.8 units.     

Quantification of trimesic acid in liver, spleen and urine by high-performance liquid chromatography coupled to a photodiode-array detection

The quantification of trimesic acid, a constitutive organic linker from the biodegradable porous iron(III) trimesate MIL-100(Fe) (MIL stands for Materials from Institut Lavoisier), has been performed in different biological complex media (liver, spleen and urine) using a liquid-liquid extraction procedure. A recovery exceeding 92 wt% was achieved from rat tissues and urine spiked with trimesic acid. After extraction, the determination of the trimesic acid concentration was realised by using a simple and accurate high-performance liquid chromatography (HPLC) method using photodiode-array detection (PDA) and aminosalicylic acid, as internal standard. Linearity of this method was kept from 0.01 to 100mg of trimesic acid per liter of urine and from 0.05 to 5.00 wt% of trimesic acid per tissue weight. The limit of detection of the method was 0.01 μg per injection. This method was finally applied to analyze and quantify the amount of trimesic acid in rat urine and tissue samples at the different stages of degradation of MIL-100(Fe).     

THE DISTRIBUTION OF GLUTAMATE DECARBOXYLASE IN RAT TISSUES; ISOTOPIC VS FLUORIMETRIC ASSAYS

—The activity of glutamate decarboxylase (GAD, EC 4.1.1.15) in normal and neoplastic rat tissues was determined by two assay methods, one based on the production of ¹⁴CO₂ from [¹⁴C]glutamic acid and the other on the fluorimetric measurement of γ-aminobutyric acid (GABA) formation. Activities obtained with the isotopic assay were high in every tissue (ranging from over 800 in liver and brain to 107nmol CO₂/min/g in lung). They were drastically diminished by Triton X-100, by an oxygen-free atmosphere or by the mitochondrial electron transport inhibitors, rotenone and antimycin A. Activities measured fluorimetrically were significant in only a few tissues and were stimulated by Triton (e.g. from 299 to 569 nmol GABA/min/g brain) but were unaffected by rotenone. For several tissues after Triton treatment the fluorimetric and isotopic assays (in air) gave the same results (i.e. the two end products, CO₂ and GABA were in stoichiometric agreement); however, the fluorimetric assay remains the more reliable measure of GAD activity since Triton may not inhibit completely the non-GAD dependent decarboxylation of glutamate in all types of tissue preparations. The hepatic, renal and mammary tumours tested were devoid of GAD; among non-neural normal tissues, kidney, liver and, possibly, adrenal gland contained significant GAD activity. In kidney and liver the activity was 15 and 10 per cent of that in brain.     

Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection

A method for the detection of ascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection and a technique for stabilization of the vitamin are described. Since less than 1 pmol of ascorbic acid can be detected, this assay provides significantly greater sensitivity than nearly all of the currently available procedures. Stabilization of 10 pmol or less of ascorbic acid at room temperature for up to 4 h and for several weeks at -70 degrees C facilitates storage of a large number of samples and measurement of ascorbic acid using an automated sampling device. This method was used to quantitate the amounts of ascorbic acid in human polymorphonuclear leukocytes and bovine adrenomedullary chromaffin granules. The calculated concentrations found for human neutrophils (1.35 mM) and bovine chromaffin granules (10.0 mM) are in agreement with previously published data. The assay is suitable for the determination of ascorbic acid in biological samples where only a small amount of tissue is available or very low amounts of ascorbic acid are found. This method is the first application of coulometric electrochemical detection to ascorbic acid HPLC analysis.