Solid phase immunoradiometric assay for porcine serum ferritin

1. A solid phase immunoradiometric assay using anti-serum coated polystyrene tubes, is described for the assay of porcine serum ferritin. 2. The mean concentration of ferritin in the serum of both male and female pigs (Sus scrofa) was 12.1 micrograms/l +/- 8.7 micrograms (range less than 1-35 micrograms/l) and no sex differences were observed in 40 pigs from 1 day to 4 years old. 3. Serum ferritin increased with increasing body iron stores in iron loaded pigs as assessed by hepatic iron concentration. 4. The assay is sensitive (detecting less than 1 microgram/l), reproducible, specific and it does not cross-react with human or rat ferritin.     

Iron stores in female blood donors evaluated by serum ferritin

Iron stores were evaluated by serum ferritin determinations in 948 menstruating and 141 non-menstruating female blood donors. Blood donation was associated with a decrease in ferritin. First-time donors (n = 163) had a geometric mean ferritin of 24 micrograms/l and multiple-time donors a value of 19 micrograms/l (p less than 0.01). In the donating population 31.5% had ferritin values less than 15 micrograms/l (i.e. depleted iron stores). Menstruating donors had lower mean serum ferritin than non-menstruating donors (p less than 0.001), and a higher frequency of ferritin values less than 15 micrograms/l (p less than 0.05). There was no relationship between ferritin levels and the number of pregnancies. The frequency of donations was more predictive of ferritin levels than the number of donations. Mean ferritin displayed a moderate fall up to the 2nd donation, and was hereafter relatively constant, whereas an increase in donation frequency was accompanied by a significant decrease in ferritin. Female donors, especially when phlebotomised greater than or equal to 3 times per year, should have their iron status checked at appropriate intervals by measurement of serum ferritin and should be advised regular iron supplementation.     

Isolation of a porcine hepatic ferritin receptor

1. A ferritin receptor has been isolated from porcine liver and has been partially purified using affinity chromatography. 2. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support which facilitates the separation of bound and free ligand. 3. An affinity constant of 2.9 x 10(9) mol-1 litre was determined for the purified hepatic ferritin receptor. 4. The molecular weight of the receptor was estimated to be approximately 53,000 by gel electrophoresis. 5. Binding of ferritin to the insolubilized receptor was unaffected by a 100-fold excess of bovine albumin, porcine and human transferrin, and human asialo-orosomucoid. 6. Binding was specific for porcine ferritin with no demonstrable binding of rat or human ferritin.     

Serum ferritin in patients with various haemolytic disorders.

174 serum ferritin assays in 121 patients with various haemolytic disorders have been performed. The mean serum ferritin levels were significantly increased in all these disorders in contrast to healthy controls. The highest serum ferritin levels were found in pyruvate kinase (PK) deficiency, moderate increase was observed in hereditary sphaerocytosis (HS) and in autoimmune haemolytic anaemia (AIHA) with massive haemolysis and in glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. Mild elevation of serum ferritin levels was depicted in paroxysmal nocturnal haemoglobinuria (PNH), in beta thalassaemia minor and in other types of haemoglobinopathies. The range of values was associated with a degree of haemolysis and its relation to duration of the disease was not apparent in most cases. Highly significant differences between serum ferritin levels in splenectomized and non-splenectomized patients with HS and between serum ferritin levels in patients with AIHA with massive haemolysis or in remission were found. As compared to normal controls, significant increase of serum ferritin levels was observed even in patients with AIHA in remission or in splenectomized patients with HS. In two patients with PK deficiency the levels exceeding 2,000 micrograms/l indicated manifest iron overload. A reliability of serum ferritin assay as an index of iron stores in haemolytic disorders has been discussed.     

Fermentation production ofL-Homoserine byCorynehacterium sp. and its possible use in the preparation of threonine and lysine

A mutant Corynebacterium sp. requiring threonine and cultivated for 3 d in a medium containing 15% sucrose, 8% corn-steep and 50 micrograms biotin per litre accumulated 14.5 g L-homoserine per litre. The possibility of fermenting the homoserine obtained for threonine and lysine production was investigated.     

Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique.

A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.     

Impaired phagocytic activity of neutrophils in patients receiving haemodialysis: the critical role of iron overload.

The metabolic burst (as measured by the spontaneous and stimulated nitroblue tetrazolium tests), the phagocytosis of heat inactivated bakers'' yeast and of Staphylococcus aureus, the killing of Staph aureus, and the myeloperoxidase activity of polymorphonuclear neutrophils were studied in 11 patients receiving maintenance haemodialysis. Of these patients, six were polytransfused and had high serum ferritin concentrations (mean 5940 (SD 2925) micrograms/l; group 1), and five had normal serum ferritin values (mean 171 (116) micrograms/l; group 2). Patients in group 1 had a history of more infectious episodes (0.167 v 0.025 per patient per month) and significantly more genitourinary infections (p = 0.015) than those in group 2. Phagocytosis and myeloperoxidase activity were severely reduced in group 1 but normal in group 2. Percentages of neutrophils ingesting one or more particles together with the index of phagocytosis in patients'' serum were inversely correlated with serum ferritin concentrations. Four patients in group 1 were treated with desferrioxamine, and after six to 18 weeks of treatment phagocytosis and myeloperoxidase activity had returned to normal in three of them. These data suggest that in patients receiving haemodialysis iron overload due to multiple transfusions plays an important part in the mechanisms underlying the susceptibility to bacterial infections, mediated at least partially through impaired neutrophil function.     

The course of serum ferritin in blood donors

In the course of 18 months there occurred a decrease in the serum ferritin concentration from 58 +/- 2 micrograms/l to 32 +/- 1 micrograms/l in male donors after 5-6 donations of 400 ml of blood each. Female permanent donors showed a constantly lowered content of an average of 15 +/- 1 micrograms/l. Female first donors attained the serum ferritin level of permanent female donors with the fourth donation, whereas it was not until the fifth donation that male first donors declined to the level of permanent donors. This decrease of serum ferritin content in blood donors points to a depletion of iron stores. This process should be counteracted by exerting an increased influence on nutritional habits supported by oral iron substitution and diminishing the frequency of donation, particularly for female blood donors.     

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.     

Examination of serum ferritin and erythrocyte ferritin--its role in the blood transfusion service.

Blood donors were examined for serum ferritin values and concentration of ferritin in the erythrocytes. The group of male and female donors without previous donations showed average values of 102.27 ng and 51.75 ng of ferritin per one ml of serum, respectively. Males with over 20 donations had 68.04 ng ferritin per one ml, females 37.14 ng of ferritin per one ml. The reduced serum ferritin values in multiple male and female donors is statistically significant. Serum ferritin values in women of the two groups are lower than those of males, the difference also being statistically significant. In male and female blood donors, irrespective of the number of donations, average values of 13.74 ag and 12.07 ag of ferritin per erythrocyte, respectively, were established. The difference in ferritin concentration in the erythrocytes between males and females is statistically insignificant. The correlation coefficient failed to demonstrate any dependence between erythrocyte ferritin concentration and concentration of ferritin in the serum. The object of serum ferritin determination in blood donors is to detect the earliest stage of storage iron deficiency in the organism. For the latter purpose, the determination of erythrocyte ferritin is ineffective.     

Radioimmunofixation of human ferritin following serum isoelectric focusing

Radioimmunofixation of human ferritin following isoelectric focusing of serum was developed to study the microheterogeneity of this protein in native serum without previous purification or concentration. This method requires only 2-10 microliter of serum and can be used with levels of ferritin as low as 10 micrograms/l. In this way, the extensive microheterogeneity of this protein was revealed, since in some cases it produced as many as 35 bands with isoelectric points in a pH range of 4.95-5.9. Very different isoelectric focusing patterns (spectrotypes) of ferritin were observed during the investigation of pathological sera. The high sensitivity of this technique makes it useful for the investigation of serum ferritin in diseases involving modifications of the metabolism of this protein.     

Turnover and tissue uptake of rabbit ferritin from rabbit plasma

Using a two-site immunoradiometric assay for rabbit liver ferritin normal NZW rabbits were found to have very low plasma ferritin concentrations (less than 4 micrograms/l). Purified preparations of rabbit liver and kidney ferritin were labelled with 125I and injected into rabbits. Clearance from plasma was extremely rapid with an initial half-life of 1-2 min as measured by immunoprecipitation of labelled ferritin. The rate of clearance was unaffected by the labelling procedure and by the method of ferritin purification. Autoradiography and organ uptake studies showed that 125I-rabbit liver ferritin was removed mainly by liver reticuloendothelial cells, although on a weight basis, spleen had the greatest radioactivity. These studies indicate that rabbit ferritin released into the circulation is promptly cleared by the RES.     

Separation and quantitation of serum beta-carotene and other carotenoids by high performance liquid chromatography

We describe a specific assay for serum provitamin A (alpha- and beta-carotene) by high pressure liquid chromatography (HPLC). The system separates alpha- and beta-carotene in 7.4 min using a C18 muBondapak, 10-micron particle size column with a mobile phase of acetonitrile-chloroform 92:8 at 2 ml per min and a 462 nm detector. The HPLC assay had a recovery of 94.8% of added beta-carotene and, at a serum concentration of 215.2 micrograms/L, had within-run and between-run precisions of 3.1% and 3.6%, respectively. In 65 subjects, the HPLC-determined provitamin A (alpha- and beta-carotene) value was 343 +/- 166 micrograms/L and averaged 23.4 +/- 7.9% (range 9-43%) of the values obtained by a traditional colorimetric assay for total serum "carotenes." Although total serum carotenes showed no relationship to serum vitamin A (r = -0.048; P = 0.78), HPLC-determined alpha- and beta-carotene was significantly inversely correlated (r = -0.357; P = 0.05).     

Synthesis of ferritin by neuroblastoma

Ferritin is an iron-containing protein which is a normal component of serum. The levels of ferritin are increased in the sera of some children with neuroblastoma, and this increase appears to be a potent indicator of prognosis. To determine whether synthesis of ferritin by the tumor cells contributes to these increased serum levels, we examined incorporation of radiolabeled leucine by CHP 126, a neuroblastoma derived cell line, into ferritin. Using sequential immunoprecipitation and gel electrophoresis of sonicates from cells maintained in medium containing iron in amounts standard for tissue culture, incorporation of label into ferritin was 0.04% of that into total protein synthesized over the same time period. Addition of up to 40 micrograms of iron as ferric ammonium citrate increased ferritin synthesis to a maximum of 0.16% without altering synthesis of total protein. The pattern of iron-induced enhancement in the neuroblastoma cells was similar to that which was seen using Chang liver cells, a cell line well known to be capable of ferritin synthesis. These results confirm that neuroblastoma cells can synthesize ferritin and that synthesis is regulated by exogenous iron.     

Endotoxin inactivating activity of rat serum

The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37 degrees C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 micrograms Escherichia coli LPS per hour. Heating serum for 45 min at 56 degrees C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 micrograms LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56 degrees C, inactivates between 10 and 15 micrograms LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 micrograms LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56 degrees C) and lead acetate reduce LPSI activity.     

Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml^–1, with a mean value of 479±286 (SD) ng ml^–1. Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml^–1 in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml^–1. There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112±0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.     

Measurement of serum ferritin by a 2-site immunoradiometric assay

A 2-site immunoradiometric assay (2-site IRMA) for human serum ferritin is carried out by reaction of the ferritin solution with a solid-phase anti(human ferritin), followed by a second reaction in which the insoluble product is incubated with purified, radioactively labeled anti(human ferritin). Unreacted labeled antibodies remain in solution and are washed away. As the amount of ferritin increases, the radioactivity in the solid-phase increases. Factors affecting the assay were evaluated including (a) concentration and stability of solid-phase antibody, (b) variation in temperature, reaction times, and reagent concentrations, (c) stability and storage of antibody coated tubes, (d) effect of tube washing cycles, (e) effect of serum proteins and anticoagulants, (f) organ specificity of ferritin.A paradoxical fall in dose-response was seen at high dose. Statistical analysis, dose interpolation, and automatic data processing were carried out by a generalization of the logit/log method and computer programs used in conventional radioimmunoassay. The dependence of the variance on the position on the dose-response curve is different than that seen in radioimmunoassay systems and is reflected in a greater effective assay range. 2-Site IRMA may also have advantages in reagent stability, specificity, antigen protection, and suitability for automation. The properties of 2-site IRMA are closely related to the usual IRMA assay system, but 2-site IRMA is more economical in antigen, is unlikely to be subject to deleterious allosteric reactions, and has a lower zero dose-response (0.5–2% of the total radioactivity in the system).     

High rate anaerobic digestion of piggery manure with polyurethane sponges as support material

Summary The use of polyurethane foam sponges to colonize methanogenic associations for the digestion of piggery manure has been investigated. Fermentors containing polyurethane pads as colonization matrix reached a biogas production rate of ca. 2.0 litres per litre reactor per day (30–33°C), hydraulic retention time 7.5 daysl and a biogas yield of 16 litres per litre piggery manure (7–9% TS). Corresponding control fermentors containing no pads reached a gas production rate of 1.3 litres per litre reactor per day and only about 10 litres biogas per litre piggery manure.     

Haem binding to ferritin and possible mechanisms of physiological iron uptake and release by ferritin.

Haem binding to horse spleen ferritin and Pseudomonas aeruginosa bacterioferritin has been studied by spectroscopic methods. A maximum of 16 haems per ferritin molecule, and 24 haems per bacterioferritin molecule, has been shown to bind. The influence of the bound haem on the rate of reductive iron release has been investigated. With a range of reductants and in the absence of haem the rate of release varied with the reductant, but in the presence of haem the rate was both independent of the reductant and faster than with any of the reductants alone. This indicates the rate-limiting step for iron release in the absence of haem was electron-transfer across the protein shell. Based on the results obtained with the in vitro assay system and from a consideration of data currently in the literature, plausible schemes for ferritin and bacterioferritin iron uptake and release are described.     

Cytidine 5'-monophospho-N-acetylneuraminic acid or a related compound is the low Mr factor from human red blood cells which induces gonococcal resistance to killing by human serum

A low-Mr factor which induces gonococcal resistance to complement-mediated serum killing has been partially purified from lysates of mixed red and buffy coat cells from human blood. The lysates were dialysed against Tris buffer for 24 h at 25 degrees C with the diffusate being continuously recycled through a column of QAE-Sephadex A25. After elution in an NaCl gradient, the active fractions were both desalted and further purified on Sephadex G10. A second fractionation on QAE-Sephadex A25 and desalting with Sephadex G10 preceded further purification by repeated high-pressure liquid chromatography (HPLC) using a DEAE anion exchange column and desalting with Sephadex G10. Less than 500 micrograms of material showing one peak in HPLC was obtained from 1 litre of blood. After NMR had indicated the possible presence of pyrimidine nucleotide, carbohydrate and N-acetyl groups, nanogram quantities of a commercial preparation of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were shown to induce gonococci to serum resistance. The synthetic CMP-NANA also co-eluted with the preparation from blood cells in HPLC, and the two materials were indistinguishable in their patterns of acid and heat lability. Furthermore, the resistance-inducing activity of both materials was inhibited by cytidine monophosphate, which is known to inhibit sialylation reactions by CMP-NANA. It appears therefore that the resistance-inducing factor is CMP-NANA or a closely related compound. If the factor is CMP-NANA, biological activities indicated that the cell lysate from 1 litre of blood contained about 40 micrograms, and the most purified preparation contained only about 1%. With this minute amount in a mixture, the presence of CMP-NANA or a closely related analogue could not be established unequivocally by NMR.