Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes

Information on the molecular distribution and ageing trend of brain iron in post‐mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H‐ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of ≈ 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L ‐Ferritin also showed an increasing trend during life although the concentrations were ≈ 50% less than that of H‐ferritin at each age point. Neuromelanin was not detectable during the first year, increased to ≈ 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high‐spin trivalent iron is probably arranged in a ferritin‐like iron?oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H‐ and L‐ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.     

The Relation of Hepcidin to Iron Disorders,Inflammation and Hemoglobin in Chronic Kidney Disease

The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by ⁵¹Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m² (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.     

Iron Requirements and Aluminum Sensitivity of an Hydroxamic Acid-Requiring Strain of Bacillus megaterium

Bacillus megaterium strain ATCC 19213 secretes a ferric-chelating secondary hydroxamic acid, whereas a mutant (strain SK11) derived from it cannot produce a hydroxamate. Strain SK11 could be cultivated in a sucrose-mineral salts medium (treated with Chelex 100 to reduce trace metals) in the absence of added hydroxamate, if the inoculum was high. The lowest iron supplements necessary for maximal growth of both strains were equivalent (0.01 to 0.04 mug of iron per ml). Addition of either aluminum (0.5 mug/ml) or chromium (0.1 mug/ml) to the medium prevented full growth of strain SK11 at the minimal iron concentration, although elevated iron (1 mug/ml) reversed this inhibition. The iron-free secondary hydroxamate, Desferal, also abolished aluminum and chromium inhibition of strain SK11, producing maximal population densities at the low iron concentration. Growth of the hydroxamate-producing strain 19213 was not altered significantly by the aluminum or chromium levels which inhibited strain SK11. However, strain 19213 responded to these metals by increasing its secretion of a secondary hydroxamate. It was concluded that aluminum and chromium interfered with iron incorporation, either directly or by formation of nonutilizable aggregates with iron. The secondary hydroxamates may have overcome this interference by solubilization of iron for delivery to a single uptake process, or the ferric-hydroxamate chelate may enter the cell by an alternate route.     

Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.     

Iron Status is Associated with Asthma and Lung Function in US Women

BackgroundAsthma and iron deficiency are common conditions. Whether iron status affects the risk of asthma is unclear.ObjectiveTo determine the relationship between iron status and asthma, lung function, and pulmonary inflammation.MethodsRelationships between measures of iron status (serum ferritin, serum soluble transferrin receptor (sTfR), and sTfR/log10ferritin (sTfR-F Index)) and asthma, lung function, and pulmonary inflammation were examined in women 20-49 years in the National Health and Nutrition Examination Survey. Logistic, linear, and quadratic regression models accounting for the survey design of NHANES were used to evaluate associations between iron status and asthma-related outcomes and were adjusted for race/ethnicity, age, smoking status, income, and BMI.ResultsApproximately 16% reported a lifetime history of asthma, 9% reported current asthma, and 5% reported a recent asthma episode/attack (n = 2906). Increased ferritin (iron stores) was associated with decreased odds of lifetime asthma, current asthma, and asthma attacks/episodes in the range of ferritin linearly correlated with iron stores (20-300ng/ml). The highest quintile of ferritin (>76 ng/ml) was also associated with a decreased odds of asthma. Ferritin levels were not associated with FEV1. Increased values of the sTfR-F Index and sTfR, indicating lower body iron and higher tissue iron need, respectively, were associated with decreased FEV1, but neither was associated with asthma. None of the iron indices were associated with FeNO.ConclusionIn US women, higher iron stores were inversely associated with asthma and lower body iron and higher tissue iron need were associated with lower lung function. Together, these findings suggest that iron status may play a role in asthma and lung function in US women.     

Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 microg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552+/-152 ng/ml) and acute stressed animals showed an intermediate level (375+/-105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153+/-109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113+/-55 ng/ml for males; 128+/-25 for females) and the non-stressed humans (before exercise) show a lower concentration (63+/-37 ng/ml for male; 60+/-20 for female), respectively.     

Iron deficiency in obese postmenopausal women

Objective: This study evaluates whether the iron deficiency suggested in children and adolescents with overweight is also present with increasing age. Research Methods and Procedures: We examined 50 consecutive postmenopausal nondiabetic white women with a BMI ≥30 kg/m² and 50 non‐obese seemingly healthy women as a control group. In addition to the traditional indices of iron status, we measured the soluble transferrin receptor (sTfR) levels, a sensitive and highly quantitative indicator of early iron deficiency not influenced by the acute phase response. Results: Obese women have higher serum sTfR levels than control subjects [1.38 (range, 0.89 to 2.39) vs. 1.16 mg/dL (range, 0.69 to 2.03 mg/dL); p < 0.001]. However, no difference in ferritin concentration was observed between the groups [70.50 (range, 18 to 219) vs. 69.50 ng/mL (range, 24 to 270 ng/mL); p = not significant]. A positive correlation between BMI and sTfR concentration was detected. On multiple regression analyses, BMI (positively) and ferritin (inversely) were independent predictors accounting for sTfR. Discussion: These results suggest that a moderate degree of iron deficiency is also present among adult women with obesity. The determination of sTfR is useful in the evaluation of iron status in this condition. Further studies with a greater number of patients are required to investigate the relationship between tissue iron concentrations and obesity.     

Changes in the tumor marker concentration in female patients with hyper-, eu-, and hypothyroidism

The levels of 6 circulating tumor markers were evaluated in a total of 131 female subjects with altered thyroid states; 36 normal subjects, 46 hyperthyroid patients with Graves' disease, and 49 primary hypothyroid patients. The mean CEA concentration was observed to be significantly higher (p less than 0.02) in hypothyroid patients than in normal and hyperthyroid patients (1.1 +/- 0.1 ng/ml, 0.8 +/- 0.1 ng/ml and 0.8 +/- 0.1 ng/ml, respectively). Similarly, the mean serum CA 125 concentration in hypothyroid patients was higher (p less than 0.02) than in normal and hyperthyroid patients (13.0 +/- 2.6 U/ml, 7.6 +/- 1.1 U/ml and 5.5 +/- 0.8 U/ml, respectively), and the mean serum CA 15-3 concentration in hypothyroid patients was significantly higher than in normal subjects (p less than 0.01) and hyperthyroid patients (p less than 0.001) (16.2 +/- 0.9 U/ml, 13.9 +/- 0.6 U/ml and 10.6 +/- 0.5 U/ml, respectively). No statistical difference was found in mean CA 19-9 in the three subject groups. AFP in the hypothyroid patients (3.6 +/- 0.3 ng/ml) was significantly higher (p less than 0.05) than in normal subjects (2.6 +/- 0.2 ng/ml) and hyperthyroid patients (1.7 +/- 0.2 ng/ml) (p less than 0.01). On the other hand, serum ferritin was low in the hypothyroid patients (65.9 8.0 ng/ml) and significantly increased (69.1 +/- 9.0 ng/ml) (p less than 0.02) with the normalization of thyroid function. In hyperthyroidism, serum ferritin (70.2 +/- 7.0 ng/ml) was significantly higher than in the hypothyroid patients (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron utilization studies in Citrobacter species

Abstract Seventy-one strains of Citrobacter were screened for iron scavenging mechanisms by biologic and chemical assays. Essentially all citrobacteria (70 / 71) were found to elaborate enterobactin-like siderophores by both biologic and chemical assays, however only C. koseri (C. diversus) was found to produce aerobactin. The concentration of ethylenediamine di( o -hydroxyphenylacetic acid) (EDDA) required to inhibit the growth of individual Citrobacter strains by depleting free iron ranged from 250 μg/ml to 1000 μg/ml. Iron utilization studies of selected Citrobacter isolates indicated that hemin and hematin could reverse the effects of iron limitation on growth under iron-stressed conditions (1000 μg/ml of EDDA). Two C. koseri strains grown under iron-restricted conditions showed similar changes in their whole cell protein profiles including induction of high molecular mass proteins (72–83 kDa) which may play a role in iron acquisition under iron-stressed conditions. The collective results support an additional virulence-associated mechanism for C. koseri strains which may help explain the greater pathogenic potential this group has for causing serious extraintestinal disease in humans.     

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushing's syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.     

Pulsatile nature of luteinizing hormone release is maintained during luteinizing hormone-releasing hormone stimulation in normal male rats

The plasma LH concentration is believed to be reasonably steady in normal male rats. We found that LH is released in a regular pulsatile fashion. The overall mean concentration of plasma LH in normal male rats was 46.6 +/- 4.4 (mean +/- SEM) ng/ml. The normal male rats showed periodic LH pulses: the mean pulse amplitude was 144.4 +/- 25.5 ng/ml and the inter-peak interval was 22.5 +/- 2.0 min. Each pulse lasted 9.7 +/- 0.8 min. When LH-RH (1 microgram/kg) was injected as a bolus, the peak concentration was attained in 10-30 min reaching a peak concentration of 279.4 +/- 39.6 ng/ml. Distinct pulsatile bursts of plasma LH were discernible during the period of elevated plasma LH concentration. When a higher dose of LH-RH (5 micrograms/kg) was administered, the LH concentration slowly increased to a peak concentration of 400.2 +/- 38.7 ng/ml in 20-40 min. The pulsatile nature of the LH concentration was recognizable with distinct bursts. We have observed that: (a) normal male rats release LH in a pulsatile fashion with an approximate 20-min inter-peak interval; (b) mean LH pulses last less than 10 min, and (c) the LH pulses are visible even with elevated LH and LH-RH concentrations in the general circulation.     

Association of Iodine and Iron with Thyroid Function

Iodine and iron are essential elements for healthy thyroid function. However, little is known about the association of iron and iodine with thyroid function in the general US population. We investigated iron and iodine status in relation to concentrations of thyroid hormones. We included 7672 participants aged 20 and older from three surveys (2007–2008, 2009–2010, and 2011–2012) of the National Health and Nutrition Examination Survey. Serum thyroid measures (including free and total T3 and T4, and TSH), serum iron concentration, and urinary iodine concentrations were measured. Multivariate linear regression models were conducted with serum thyroid measures as dependent variables and combinations of serum iron concentration and urinary iodine concentration as predictors with covariate adjustment. Logistic regression models were performed with TSH levels (low, normal, and high) and combinations of serum iron concentration and urinary iodine concentration. Overall, 10.9% of the study population had low iron; 32.2 and 18.8% had low or high iodine levels, respectively. Compared with normal levels of iron and iodine, normal iron and high iodine were associated with reduced free T3 and increased risk of abnormal high TSH. Combined low iron and low iodine was associated with reduced free T3 and increased TSH. In addition, high iodine was associated with increased risk of abnormal high TSH in females but not in males. Thyroid function may be disrupted by low levels of iron or abnormal iodine, and relationships are complex and sex-specific. Large prospective studies are needed to understand the mechanisms by which iron interacts with iodine on thyroid function.     

Iron metabolism during lactation and suckling in a marsupial, the quokka (Setonix brachyurus)

The iron status and transfer of iron through the milk during lactation were determined in a marsupial, the quokka, Setonix brachyurus. Lactating animals were not anaemic and had similar liver and spleen non-haem iron values to non-lactating female adult animals but about 40% less non-haem iron than male adults. The milk iron concentration was very high during the period of lactation when the young is confined to the pouch, averaging about 20 micrograms/ml (eight times the plasma iron concentration), but fell markedly at the time when the young commence to leave the pouch. Blood haemoglobin concentration increased during pouch life of the young to reach adult levels at about 180 days, and liver non-haem iron concentration increased during the same period to values nearly 20 times greater than in their mothers. After the young left the pouch the non-haem iron concentration fell rapidly to the adult level while haemoglobin concentrations were maintained. It is concluded that milk is an adequate source of iron during pouch life of the quokka and enables the animal to build up iron stores which can be utilized after leaving the pouch.     

Decorporation of Iron Metal Using Dialdehyde Cellulose-Deferoxamine Microcarrier

Deferoxamine iron chelator has a limited therapeutic index due to rapid clearance from blood and possesses dose-limiting toxicity. Therefore, an intravenous deferoxamine delivery system based on dialdehyde cellulose (DAC) polymer was developed and its efficacy and toxicity were tested in iron-overloaded animals. The amino groups of deferoxamine were conjugated to free aldehyde moieties of dialdehyde cellulose via Schiff base reaction to form dialdehyde cellulose-deferoxamine (DAC-DFO) conjugate and characterized by Fourier transform infrared spectrophotometer, scanning electron microscope, and X-ray diffraction. The toxicity of prepared formulation was analyzed by XTT cell viability assay and LD50 study in mice. The change in serum iron levels, after intravenous administration of formulation, was observed in iron-overloaded rats. The DAC-DFO conjugate was tagged with Tc-99m to study the blood kinetics and observe change in blood circulation time. DAC-DFO conjugate was dispersible in water at concentration ～75 mg/ml. In vitro cytotoxicity assay and LD₅₀ study in mice indicated significantly enhanced safety of covalently bound deferoxamine (at >1000 mg/kg body weight compared to free drug at ～270 mg/kg dose). A preliminary scintigraphy imaging and blood clearance study, with technetium-99m, indicated prolonged circulation of conjugated DFO in rabbit blood. A single dose of formulation injected into iron overloaded animals was found to maintain the normal serum iron levels until 10 days. The polymeric conjugate was effective in maintaining normal serum iron levels until 10 days at a dose of 100 mg/kg body weight.     

Total and free testosterone in plasma of hypo- and agonadal men.

Plasma total testosterone (T), apparently free T and testosterone binding globulin (TeBG) capacity determined in 14 normal men aged 30-40 years were 461 +/- 100 ng/100 ml, 9.4 +/- 3.0 ng/100 ml and 5.7 +/- 1.9 X 10(-8) M, respectively, whereas in 16 hypogonadal men the corresponding values were 38.6 +/- 27.2 ng/100 ml, 0.47 +/- 0.41 ng/100 ml and 10.4 +/- 3.4 X 10(-8) M showing the TeBG capacity significantly higher (p less than 0.001) in hypogonadal than in normal men. Treatment of 5 hypogonadal subjects with 250 mg testosterone enanthate plus 50 mg testosterone propionate decreased (p less than 0.001) the TeBG level from 14.7 +/- 2.5 X 10(-8YM to 8.3 +/- 1.4 X 10(-8) M on day 8 after a single injection. According to this difference in TeBG, the free T fraction in plasma rose from 0.94% to 1.9% of the total T concentration. These results suggest that alteration of total plasma T affected the TeBG capacity. Decreased T levels raised and increased T concentrations suppressed TeBG, but with a delayed response to the changed T concentrations. The initial mean values in 12 patients with prostatic cancer aged 60-74 years were 397 +/- 165 ng/100 ml, 4.05 +/- 1.8 ng/100 ml and 11.9 +/- 3.3 X 10(-8) M, respectively. The TeBG capacity in these patients was significantly higher and the free T concentration significantly lower (p less than 0.001) than those of the younger normal males. After treatment with 12 g diethylstilbestrol diphosphate and orchidectomy, the TeBG increased to 33.3 +/- 13.1 X 10(-8) M and the plasma free T concentration decreased to the minimal value of 0.053 +/- 0.04 ng/100 ml.     

Elevated Systemic Hepcidin and Iron Depletion in Obese Premenopausal Females

Hepcidin, the body's main regulator of systemic iron homeostasis, is upregulated in response to inflammation and is thought to play a role in the manifestation of iron deficiency (ID) observed in obese populations. We determined systemic hepcidin levels and its association with body mass, inflammation, erythropoiesis, and iron status in premenopausal obese and nonobese women (n = 20/group) matched for hemoglobin (Hb). The obese participants also had liver and abdominal visceral and subcutaneous adipose tissue assessed for tissue iron accumulation and hepcidin mRNA expression. Despite similar Hb levels, the obese women had significantly higher serum hepcidin (88.02 vs. 9.70 ng/ml; P < 0.0001) and serum transferrin receptor (sTfR) (P = 0.001) compared to nonobese. In the obese women hepcidin was not correlated with serum iron (r = ?0.02), transferrin saturation (Tsat) (r = 0.17) or sTfR (r = ?0.12); in the nonobese it was significantly positively correlated with Tsat (r = 0.70) and serum iron (r = 0.58), and inversely with sTfR (r = ?0.63). Detectable iron accumulation in the liver and abdominal adipose tissue of the obese women was minimal. Liver hepcidin mRNA expression was ～700 times greater than adipose tissue production and highly correlated with circulating hepcidin levels (r = 0.61). Serum hepcidin is elevated in obese women despite iron depletion, suggesting that it is responding to inflammation rather than iron status. The source of excess hepcidin appears to be the liver and not adipose tissue. The ID of obesity is predominantly a condition of a true body iron deficit rather than maldistribution of iron due to inflammation. However, these findings suggest inflammation may perpetuate this condition by hepcidin‐mediated inhibition of dietary iron absorption.     

Effect of Iron on Erythropoietin Production in Anaemic Piglets

In the present study the response of plasma erythropoietin to iron injection in anaemic piglets was examined. At the age of 16 days, 4 piglets from the same litter were given 180 mg iron subcutane-ously. After iron injection, blood samples for estimation of erythropoietin activity in plasma, haemoglobin concentration, and reticulocyte counts were taken every 6 or 12 h for 3 Vi days. Plasma erythropoietin activity was estimated by a monoclonal enzyme-im-munoassay (ELISA), developed for human erythropoietin. On the day of iron injection, haemoglobin concentration ranged between 41 and 48 g/1, reticulocyte counts from 9 to 17 percent, and plasma erythropoietin between 22 and 144 mU/ml. In 3 of the 4 piglets, iron injection resulted in a 2-6 fold increase in erythropoietin activity. Maximal eryth-ropoietin activities were observed 24-42 h after injection, and after 66 h, the activities were close to the pretreatment values. It is concluded that in our experiment, iron, per se, has stimulated erythropoietin production.     

Influence of Temperature on the Iron Metabolism of a Fluorescent Pseudomonad

The iron requirement for maximal cell yields of a fluorescent pseudomonad increases as the temperature of incubation is increased. On a succinate salts medium, maximal cell yields are attained at iron concentrations of 0.10 mug/ml of added iron at 20 C and at 3.0 mug/ml of added iron at 28 C. This bacterium does not grow in the basal medium at 31 C even in the presence of 0.01 to 10 mug/ml of added iron. The inability to grow at the higher temperature is due to the loss, by this organism, of its ability to biosynthesize hydroxamate iron transport compounds at temperatures of 28 C and above, since supplementation with such compounds produced by this organism at lower temperatures promoted growth at 31 C. The biosynthesis of these compounds at lower temperatures contributes to the efficient utilization of iron by the bacterium.     

III. Serum and sputum immunoglobulin E levels in respiratory diseases in adults

Using a solid-phase radioimmunoassay, serum IgE level was determined in 46 normal subjects, 53 patients with bronchial asthma, 44 patients with chronic bronchitis and / or emphysema, and 19 patients with restrictive lung disease. Sputum IgE was measured simultaneously in 51 of the subjects. The range of serum IgE concentration in the normal subjects was wide. It varied between 15 and 750 ng/ml with a mean of 135 ng. Asthmatic patients had significantly higher levels of serum IgE with a mean of 579 ng/ml, but only 30% fell outside the normal 95% confidence limits. Patients with chronic bronchitis, emphysema and restrictive lung diseases had normal IgE levels. There was a significant correlation between serum and sputum IgE levels.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.