Enzyme‐linked immunosorbent assay to quantitate serum ferritin in the northern fur seal (Callorhinus ursinus)

Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species, and is more reliable than serum iron or total iron‐binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess (including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration). Iron overload has been documented to result in hemochromatosis in captive northern fur seals (Callorhinus ursinus); therefore, we developed an enzyme‐linked immunosorbent assay (ELISA) to measure serum ferritin in this species. The assay uses two murine anti‐canine ferritin monoclonal antibodies in a sandwich arrangement that was originally used in an ELISA to measure serum ferritin in dogs. Ferritin isolated from fur seal liver was used as a standard. Ferritin standards were linear from 0 to 50 ng/ml. Recovery of purified ferritin from fur seal serum varied from 89% to 99%. The within‐assay variability was 6%, and the assay‐to‐assay variability for two different samples was 10% and 16%. Zoo Biol 23:79‐84, 2004.© 2004 Wiley‐Liss, Inc.     

Serum indices of body stores of iron in northern fur seals (Callorhinus ursinus) and their relationship to hemochromatosis

Iron storage disease (hemochromatosis) has been reported in many species of both captive and free‐ranging animals. In this study we examined the relationship between this disease and concentrations of iron analytes in aquarium‐held northern fur seals (Callorhinus ursinus). Sera were analyzed for iron, total iron‐binding capacity (TIBC), ferritin, ceruloplasmin, and haptoglobin concentrations in a retrospective study that included samples taken over a 14‐year period. The animals ranged in age from <1 year to an estimated 23 years. Serum ferritin was measured using an enzyme‐linked immunosorbent assay (ELISA) for canine sera. The results from this assay are the first reported for any pinniped. Serum iron concentrations in presumed healthy animals ranged from 37 to 196 µg/dl, and TIBC ranged from 136 to 484 µg/dl. The transferrin saturation percentage differed significantly between male (41%) and female (63%) adult fur seals, as did the ferritin levels (54 ng/ml for males vs. 500 ng/ml for females). There was a trend toward increased serum ferritin and percent transferrin saturation with age, especially in females. The data also showed a relationship between serum iron and transferrin saturation among eight mother–pup pairs, which suggests that pups may develop increased iron levels due to placental transfer of iron and/or transfer of iron through the milk from iron‐overloaded females. Diet was considered as a factor in the development of hemochromatosis in at least three geriatric female northern fur seals, and their diets were analyzed for iron concentrations. On the basis of these results, the diets were altered by replacing a portion of the high‐iron‐content fish (herring) with a lower‐iron‐content item (squid), and discontinuing iron and vitamin C supplementation (via a multivitamin tablet). Sera were analyzed before, and 1 and 4 years after the dietary changes were implemented. Paired t‐tests showed no significant changes in the iron analytes from pre‐ to post‐diet‐change samples, which indicates that it may be too late to affect iron levels by diet alone in older animals with a chronic history of elevated iron levels. Zoo Biol 23:205–218, 2004. © 2004 Wiley‐Liss, Inc.     

Characterization of autoantibodies to ferritin in canine serum

Ferritin-binding proteins circulating in mammalian blood are thought to be involved in the clearance of ferritin. The present study characterizes canine serum autoantibodies (IgM and IgA) that react with ferritin. Canine IgM and IgA bound to bovine spleen ferritin as well as canine liver ferritin. To examine the specificity of canine IgM and IgA to ferritin H and L subunits, we used canine heart ferritin and canine liver ferritin with H/L subunit ratios of 3.69 and 0.43, respectively. Canine IgM and IgA recognized both of the H- and L-subunit-rich isoferritins, showing that their binding activities to ferritin depend on the H-subunit content. Recombinant bovine H-chain ferritin homopolymer expressed in a baculovirus expression system bound more with IgM and IgA than the recombinant L-chain homopolymer expressed under the same conditions. These results suggest that canine IgM and IgA recognize H-subunit-rich isoferritins, and that H-subunit-rich isoferritins are cleared from the circulation more rapidly than L-subunit-rich isoferritins.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Purification of fusion ferritin from recombinant E. coli using two-step sonications

Fusion ferritin, combined by heavy chain ferritin (21 kDa) and light chain ferritin (19 kDa), was expressed in recombinant E. coli. The fusion ferritin was easily purified by two-step sonications as well as gel filtration chromatography. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 Fe³⁺ mg mg^–1 of protein and 66%. Those values are larger than the previous ones of 0.2 Fe³⁺ mg mg^–1 (Kim et al. 2001).     

Characterization of ferritin and ferritin-binding proteins in canine serum

Ferritin and ferritin-binding proteins in canine serum were characterized. A certain percentage of ferritin in canine serum, but no tissue ferritin, was precipitated by centrifugation at 16,000×g for 30 min. The precipitated ferritin was found to contain two subunits corresponding to the H and L subunits of canine liver ferritin by immunoblotting, the H subunit being predominant. More ferritin was precipitated from canine sera which had been incubated with anti-rat liver ferritin antibody than from untreated sera, and the H chain also predominated. To evaluate the possibility that the autoantibody was responsible for the precipitation of canine serum ferritin, the ferritin-binding activities of canine antibodies were examined using liver ferritin-coated microtiter plates and alkaline phosphatase-labeled antibodies specific for canine IgM, IgA, and IgG heavy chains. The results showed that IgM and IgA, but not IgG, had considerable ferritin-binding activities. Given these results, we suggest that there is H-chain-rich isoferritin in canine serum, and that ferritin exists as an immune complex.     

Feeding the fairy shrimp Streptocephalus (Anostraca - Crustacea) with the rotifer Anuraeopsis

Laboratory-cultured Streptocephalus torvicornis were offered 8 concentrations (from 6 to 800 ind. ml^–1) of Anuraeopsis fissa for periods of 2 h 30 min. Two size classes, small (male: 14.7 mm± 1.6, female: 15.4 mm± 1.3) and large (male: 20.0 mm±2.0, female: 23.1 mm± 1.5), of S. torvicornis were used. Functional response for large S. torvicornis (both sexes) plateaued at 400 rotifers ml^–1, while in small specimens it did so at 200 prey ml^–1. Females consumed significantly more (30%) prey than males. Large males consumed maximum 4730 rotifers h^–1, females 6560 h^–1.     

Comparison of in vitro cytotoxicity of Fusarium mycotoxins,deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines

Three human epithelial cell lines (CaCo-2, HEp-2 and HeLa) implicated as potential targets for three Fusarium toxins were tested for the extent of survival on exposure to increasing toxin concentration and incubation periods. Cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was carried out with deoxynivalenol (DON), T-2 toxins and zearalenone (ZON) on CaCo-2, HEp-2 and HeLa cell lines. Of the three cell lines used, HeLa was the most sensitive, eliciting cell death after 2 days exposure at 100 ng ml^–1with T-2 toxin. HeLa was the only cell line to exhibit cytotoxicity towards ZON showing cell death at 1000 ng ml^–1after 2 days which increased to 4 days, showing substantial cell death at 200 ng ml^–1. HEp-2 was sensitive to DON showing cell death after 2 days (100 ng ml^–1) with complete cell death occurring at 200 ng ml^–1 after 4 days of exposure. Substantial cytoxicity of T-2 towards HEp-2 occurred after 2 days at 1000 ng ml^–1 and complete cell death occurred with 100 ng ml^–1 at day 4. The CaCo-2 cell line was generally resistant to the mycotoxins tested between 100 and 1000 ng ml^–1. This study shows that cytotoxicity of Fusarium toxins to epithelium cell lines is concentration- and time- dependant and results from ZON–HeLa interaction indicate possible cell type-mycotoxin specificity.     

The relationship between total non-haem,ferritin and haemosiderin iron in larvae of Southern Hemisphere lampreys (Geotria australis andMordacia mordax)

Summary The major iron binding protein (IBP) of larvalM. mordax has an estimated molecular weight (354,000), subunit molecular weight (18,000) and pI (5.1) identical to those recorded previously for larvalG. australis. The IBP in larvalG. australis has also been shown to be relatively heat stable and to react immunologically with antihorse spleen ferritin. The weight of total non-haem iron in the whole body, and both the ferritin and haemosiderin iron components, increased with increasing body weight in larvalG. australis. While the concentration of ferritin iron remained similar throughout larval life, the concentration of total non-haem iron and haemosiderin iron increased rapidly in animals up to a body weight of 0.1–0.2 g, but thereafter rose only slowly throughout the rest of larval life. This implies that any iron in excess of the amount required for the maintenance of a constant ferritin concentration is converted into haemosiderin iron, and that once non-haem iron has reached a particular concentration (c. 500–600 g g^–1), the rate of iron accumulation is greatly reduced. While the larvae of bothG. australis andM. mordax had very high plasma iron levels (>19,000 g 100 ml^–1), the former had significantly greater concentrations of iron in the whole body (702vs. 267 g g^–1) and more particularly in the nephric fold (7382vs. 224 g g^–1). A greater reservoir of non-haem iron could facilitate the maintenance of the large amounts of haem and erythrocytic ferritin present in this species as a result of an exceptionally high haemoglobin concentration and red blood cell number. The greater concentration of non-haem iron in the intestine ofM. mordax than ofG. australis (1338vs. 824 g g^–1), when considered in conjunction with histological studies, indicates thatMordacia mordax eliminates a larger amount of iron during the extrusion of its intestinal columnar cells.Abbreviation IBP iron binding protein     

Ecological problems of Lake Ladoga: causes and solutions

We studied the outcome of competition between a large (Brachionus calyciflorus) and a small (Anuraeopsis fissa) rotifer species at five algal (Scenedesmus acutus) concentrations (0.5 × 10⁶ to 40.5 × 10⁶ cells ml^–1) and with varying initial densities in mixed populations (100 to 0% of B. calcyciflorus or A. fissa), the combined initial biomass being 0.2 µg ml^–1 in all test jars. Experiments were conducted at 28 ± 1 °C.Regardless of food concentration, B. calcyciflorus showed a greater increase in biomass than A. fissa, peak densities (mean ± standard error) at the lowest food concentration in the controls being 1.34 ± 0.31 µg dry weight ml^–1 and 0.82 ± 0.08 dry weight ml^–1, respectively. At the lower food concentrations, A. fissa displaced B. calyciflorus and vice versa at the higher food concentrations. At the intermediate food concentrations of 4.5 × 10⁶ cells ml^–1, B. calyciflorus outcompeted A. fissa only if its initial population density was three times higher. The rates of population growth in controls varied from 0.792 ± 0.06 d^–1 to 1.492 ± 0.13 d^–1 for B. calyciflorus and 0.445 ± 0.04 to 0.885 ± 0.01 for A. fissa depending on food level. When both species were introduced together, low food levels favoured higher abundance of A. fissa than B. calyciflorus, suggesting, in nature, it is likely that small Anuraeopsis colonize oligotrophic water bodies more successfully than larger Brachionus. The results also suggest that the outcome of competition depends not only on the size of the competing species and food availability but also on their colonizing density.     

Relative importance of the trophic and direct pathways on PCB contamination in the rotifer species Brachionus calyciflorus (Pallas)

To determine the contribution of food ingestion (trophic pathway) to PCB contamination of zooplankton in the river Meuse (Belgium), we used ¹⁴C-labelled algae (Dictyosphaerium ehrenbergianum) to measure ingestion and assimilation rates in the rotifer species Brachionus calyciflorus. When the concentration of algae in the culture medium varied from 20 10³ to 200 10³ algal cells ml^–1 (0.12 to 1.18 mg Cl^–1), the Brachionus calyciflorus ingestion rate varied from 0.25 ± 0.12 to 1.52 ± 0.43 ng C ind^–1 h^–1 at 15 °C and from 0.74 ± 0.17 to 5.93 ± 0.61 ng C ind^–1 h^–1 at 20 °C. The assimilation efficiency (ratio of the assimilation rate to the ingestion rate) measured in a culture medium containing 200 10³ algal cells ml^–1 was 55.7 ± 5.8%. Since the PCB concentration measured in the phytoplankton of the river Meuse is about 3 µg PCBs g^–1 D.W., the estimated PCB contamination of zooplankton ascribable to the trophic pathway ranges from 0.22 ± 0.17 to 1.31 ± 0.77 µg PCBs g^–1 D.W. at 15 °C and from 0.64 ± 0.34 to 5.10 ± 2.10 µg PCBs g^–1 D.W. at 20°C. The lower figure based on measurements effected at 20 °C is comparable to the actual level measured in zooplankton samples collected in the river Meuse (0.69 ± 0.20 µg PCBs g^–1 D.W.). The applicability of the formula used in our estimate was checked in a 48-hour in vitro experiment in which the rotifers were fed contaminated algae. The PCB accumulation measured in the rotifers was found to coincide with the calculated PCB contamination. Additional experiments were carried out to determine the contribution of the direct pathway to PCB contamination of zooplankton living in the river Meuse (0.02 µg PCBs l^–1 of water; average dissolved organic matter: 3 mg C 1^–1). The PCB concentration in zooplankton resulting from direct uptake of PCBs from the water was estimated at 0.19 ± 0.05 µg PCBs g^–1 D.W. These results show that in zooplankton living in polluted ecosystems, PCBs are likely to accumulate via the trophic pathway to concentrations up to 30 times higher than by direct contamination. Furthermore, our estimates of PCB contamination via the trophic pathway coincide quite well with actual concentrations measured in situ.     

Iron stores at birth in a full-term normal birth weight birth cohort with a low level of inflammation

Iron stores at birth are essential to meet iron needs during the first 4–6 months of life. The present study aimed to investigate iron stores in normal birth weight, healthy, term neonates. Umbilical cord blood samples were collected from apparently normal singleton vaginal deliveries (n=854). Subjects were screened and excluded if C-reactive protein (CRP) > 5 mg/l or α1-acid glycoprotein (AGP) > 1 g/l, preterm (<37 complete weeks), term < 2500g or term > 4000g. In total, 762 samples were included in the study. Serum ferritin, soluble transferrin receptor (sTfR), hepcidin, and erythropoietin (EPO) were measured in umbilical cord blood samples; total body iron (TBI) (mg/kg) was calculated using sTfR and ferritin concentrations. A total of 19.8% newborns were iron deficient (ferritin 35 μg/l) and an additional 46.6% had insufficient iron stores (ferritin < 76 μg/l). There was a positive association between serum ferritin and sTfR, hepcidin, and EPO. Gestational age was positively associated with ferritin, sTfR, EPO, and hepcidin. In conclusion, we demonstrate a high prevalence of insufficient iron stores in a Chinese birth cohort. The value of cord sTfR and TBI in the assessment of iron status in the newborn is questionable, and reference ranges need to be established.     

Demography and population growth of Asplanchna girodi (Rotifera) as a function of prey (Anuraeopsis fissa) density

Laboratory studies on population growth and life table demography of Asplanchna girodi were conducted at 25±1 °c using Anuraeopsis fissa as prey at four (250, 500, 1000 and 2000 ind ml^–1) densities. A prey density of 100 ind ml^–1 per predator per day did not support A. girodi, while at the highest prey concentration, A. girodi reached a peak of 115±7 ind ml^–1. The age distribution of A. girodi indicated that non-adults constituted about 2/3rd of the population at all prey concentrations. A decrease in prey availability resulted in increased mortality of non-adults. At the highest prey density, the rate of population increase (r) was 1.51 d^–1. The significance of estimating mortality in population growth studies is discussed.Life table demography of A. girodi was also studied using the same prey at the same concentrations. None of the survivorship parameters (e.g. mean lifespan and mean survivorship) showed a significant relation to prey density. Net reproductive rate and generation time (but not rate of population increase) were affected by prey abundance.     

Inhibitory effect of garlic on bacterial pathogens from spices

An unconventional technique for primary screening of bacterial susceptibility to garlic (Allium sativum Linn.), using a slice from its clove, was described. Aqueous extracts of garlic were found to possess a potent bacteriostatic principle against Gram-positive as well as Gram-negative foodborne bacterial pathogens. In agar medium, the minimum inhibitory concentrations (MICs) of garlic were 6–10 mg ml^–1 for Bacillus cereus, 30–40 mg ml^–1 for Staphylococcus aureus (excepting the isolate from garlic, where the MIC was 100 mg ml^–1), 20–30 mg ml^–1 for Clostridium perfringens, 10 mg ml^–1 for Escherichia coli (30 mg ml^–1 for the garlic isolate), 40–100 mg ml^–1 for Salmonella, and 10–40 mg ml^–1 for Shigella. It inhibited the growth of all these strains, which were resistant to some commonly used antibiotics. Most of the tested strains were resistant to penicillins, although sensitive to garlic. While the growth of B. cereus and Cl. perfringens was completely inhibited at 10 and 70 mg garlic, respectively, ml^–1 test broth, their respective enterotoxin production ceased at 10 and 50 mg garlic ml^–1.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

Population growth of some genera of cladocerans (Cladocera) in relation to algal food (Chlorella vulgaris) levels

We studied the patterns of population growth of 7 cladoceran species (Alona rectangula, Ceriodaphnia dubia, Daphnia laevis, Diaphanosoma brachyurum, Moina macrocopa, Scapholeberis kingi and Simocephalus vetulus) using 6 algal densities, viz. 0.05×10⁶, 0.1×10⁶, 0.2×10⁶, 0.4×10⁶, 0.8×10⁶ and 1.6×10⁶ cells ml^–1, of Chlorella vulgaris for 18 – 30 days. In terms of carbon content these algal concentrations corresponded to 0.29, 0.58, 1.16, 2.33, 4.65 and 9.31 g ml^–1, respectively. Cladocerans in the tested range of algal levels responded similarly, in that increasing the food concentrations resulted in higher numerical abundance and population growth rates (r). The peak population densities were (mean±standard error) 71±5; 17.1±0.4, 3.6±0.3, 12.7±1.1, 18.2±2.7, 15.8±1.0 and 10.9±0.02 ind. ml^–1, respectively for A. rectangula, C. dubia, D. laevis, D. brachyurum, M. macrocopa, S. kingi and S. vetulus. In general, the lowest r values were obtained for D. laevis (0.01±0.001) at 0.05×10⁶ cells ml^–1 food level while the highest was 0.283±0.004 for A. rectangula at 1.6×10⁶ cells ml^–1 of Chlorella. When the data of peak population density for each cladoceran species were plotted against the body length, we found an inverse relation, broadly curvilinear in shape. From regression equations between the food level and rate of population increase, we calculated the theoretical food quantity (the threshold level) required to maintain a zero population growth (r = 0) for each cladoceran species, which varied from 0.107 to 0.289 g ml^–1 d^–1 depending on the body size. When we plotted the cladoceran body size against the corresponding threshold food levels, we obtained a normal distribution curve. From this it became evident that for up to 1300 m body size, the threshold food level increased with increasing body size; however, beyond this, the threshold level decreased supporting earlier observations on rotifers and large cladocerans.     

DNA buoyant densities ofTrypanosoma (Schizotrypanum) species from bats in Ontario,Canada

Summary Two species of trypanosomes,Trypanosoma hedricki andT. myoti, isolated respectively fromEptesicus fuscus andMyotis lucifugus, were cloned.T. hedricki produced long trypomastigotesin vitro (mean length 22.1±0.3 m); the buoyant densities of its kinetoplastic and nuclear deoxyribonucleic acid (kDNA and nDNA) were respectively 1.700 and 1.710 g ml^–1 and it possessed a small amount of a satellite component (C-DNA) of density 1.716 g ml^–1. This species is in this respect closely related toT. dionisii. T. myoti produced long trypomastigotesin vitro (mean length 20.1±0.2 m); its DNA buoyant densities (g ml^–1) were: kDNA 1.700 and nDNA 1.710 (no C-DNA was detected). In this respect, therefore,T. myoti is closer toT. cruzi than the other bat trypanosomes. ac]19830129     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Photochemical mobilization of ferritin iron

The release of Fe from horse spleen ferritin through photochemical reduction of Fe³⁺ to Fe²⁺ was studied in vitro. Spectrophotometric measurement of the Fe(Ferrozine)₃ ^4– complex (specific for Fe²⁺) was used to quantify rates of Fe²⁺ mobilization. Light radiation from cool white fluorescent plus incandescent bulbs effectively promoted the rate of Fe²⁺ release. Compounds known to be present in plants provided further regulation of photorelease. Reductive removal from ferritin was inhibited by phosphate, and hydroxide, whereas citrate, oxalate, tartrate, and caffeate enhanced the release. Of the organic acids studied, caffeate was the only compound which induced detectable Fe²⁺ mobilization in the absence of irradiation. Rate constants for photorelease ranged from 2.7×10^–3 sec^–1 (pH=4.6) to 2.1×10^–3 sec^–1 (pH=7.1) at 26.5°C. These findings provide one possible explanation for the low level of ferritin-Fe in healthy, illuminated plant tissue.