Assessing and influencing the fractional contribution of erythrocyte-bound 59Fe to individual 59Fe tissue content in murine 59Fe distribution studies.

BACKGROUND: Murine proteins of iron homoeostasis are frequently manipulated to investigate the mechanisms of iron-distribution and their toxicological consequences. Beyond subtracting erythrocyte-bound 59Fe of the residual blood content determined for each tissue (subtraction method), procedures are needed to determine 59Fe distribution in murine models of, e.g. inflammation or diabetes that cause local hyperaemia and changes in microcirculation. AIM: Two new methods were developed to correct total 59Fe tissue content individually for erythrocyte-bound 59Fe-labelled haem iron. METHODS: Iron-deficiency and iron-overload was induced in male C57BL6 mice by feeding of respective diets. Distribution of 59Fe between different tissues was determined 24h, 14, and 28 days after intravenous injection of 59Fe trace amounts. Haem-bound 59Fe was separated from non-haem 59Fe in homogenates from all tissues by dispersion in a mix of lipophilic cyclohexanone and hydrophilic H3PO4 (separation method). Moreover, the reduction of 59Fe-labelled tissue blood content was determined in all organs after in vivo saline perfusion via the left ventricle (perfusion method). RESULTS AND DISCUSSION: 59Fe-labelled non-haem iron determined by the separation method was not significantly different from values determined by the subtraction method, except for the iron-deficient spleen 14 and 28 days after 59Fe injection when the separation method yielded approximately 20% higher values. Approximately 20% of 59Fe-labelled haem iron spilled over into the hydrophilic phase. The impact of this error decreases in parallel to 59Fe radioactivity in the residual tissue blood content: thus, it is higher in iron-deficient mice which accumulate more 59Fe in their erythrocytes than iron-adequate and iron-rich mice. For the same reason this type of error is more marked after long distribution periods and in organs with high residual blood content. Saline perfusion via the left ventricle reduced total blood content in mice to less than 10%. Liver (95%) and duodenum (94%) showed the highest removal of blood while it is lowest in spleen (66%) and lungs (69%). CONCLUSIONS: The separation and the perfusion method can be used to correct the impact of erythrocyte-bound haem iron individually. A margin of error below 10% was determined for all organs except for spleen, lungs, and fat. Both methods can be applied sequentially to obtain satisfactory results in spleen, lungs, and fat.

A method to assess 59Fe in residual tissue blood content in mice and its use to correct 59Fe-distribution kinetics accordingly.

BACKGROUND: Dysregulation of body iron-distribution may induce oxidative damage. To investigate the molecular mechanisms of iron homeostasis, corresponding essential genes are manipulated by many working groups. This asks for a simple method to pursue the resulting impact on body iron-distribution in mice. AIM: To develop a method for the assessment of (59)Fe in residual tissue blood content and to correct this influence in (59)Fe body distribution studies. METHODS: Iron status in male adult C57BL6 mice was adjusted by feeding diets with different iron content. Fractional contribution of organs to total body weight was determined after dissection. (59)Fe-labelled blood was injected in recipient mice to estimate total blood volume and residual blood content in all organs and tissues. The main experiment used these data to correct total (59)Fe tissue content at six intervals after (59)Fe injection (12h-28 days). RESULTS AND DISCUSSION: The sum of (59)Fe in all organs was the same as determined in each mouse before dissection. (59)Fe in whole blood remained constant from the 4th day after injection on, and was highest in iron-deficiency. As in other species, residual blood content was highest in spleen and lungs. Nevertheless, (59)Fe in the iron-deficient spleen decreased to zero and intestinal (59)Fe-content also decreased significantly over time after correction for (59)Fe in residual blood. These findings suggest correct assessment of compartment sizes and (59)Fe in residual blood in each organ. CONCLUSIONS: Differences in (59)Fe-distribution between iron status reflected changes in the expression of proteins of iron-transport and its regulation correctly. Thus, the method seems suitable to analyse body iron-distribution in consequence to genetic manipulations of murine iron homeostasis which is a prerequisite to assess possible toxicological consequences of iron supplementation.

The role of meat to improve the critical iron balance during weaning.

BACKGROUND: Iron requirements during the weaning period are the highest per unit body weight during human life, and diet is often insufficient to cover iron needs. For the first time in infant nutrition the absorption of both nonheme and heme iron from a typical weaning gruel after addition of meat with and without ascorbic acid (AA) to improve bioavailability was studied. METHODS: Nonheme and heme iron absorption from gruel was measured in 33 adults using 2 radioiron isotopes--an inorganic iron salt to label nonheme iron, the other biosynthetically labeled rabbit hemoglobin to label heme iron. Iron absorption was measured from the basal gruel (based on milkpowder and cereals) and from basal gruel added 20 g red powdered meat, alone and together with 20 mg AA in 4 different trials. RESULTS: Nonheme iron absorption from the basal meal was 0.33 mg/1000 kcal and the increase from added 20 mg AA was 39%, whereas addition of red meat increased nonheme iron absorption by 85%. This latter increase was unexpectedly high. Total iron absorption was further increased by heme iron absorption of 0.23 mg Fe/1000 kcal. When adding both meat and AA, total iron absorption amounted to 1.08 mg iron/1000 kcal, ie, exceeding 1 mg/1000 kcal, a level estimated to correspond with daily iron requirements in 95% of infants aged 12 months. CONCLUSIONS: Addition of powdered red meat to weaning gruels markedly increased total iron absorption. A weaning diet with added powdered meat and AA may serve as a viable option to satisfy the body's high iron requirements during this critical period.

A simplified method for determination of radioactive iron in whole-blood samples.

For studies on iron absorption in man radioisotopes represent an easy and simple tool However, measurement of the orbital electron emitting radioiron, 55Fe, in blood is difficult and insufficiently described in the literature. The present study describes a relatively simple method for simultaneous determination of 55Fe and 59Fe in blood, using a dry-ashing procedure and recrystallization of the remaining iron. The detection limit of the method permits measurements of 0.1 Bq/ml blood thus allowing detection of less than 1% absorption from a 40 kBq dose, which is ethically acceptable in humans. The overall recovery of radioiron from blood is more than 90%, and the coefficient of variation, as judged by the variation in the ratio 55Fe/59Fe is in the order of 4%. Combined with whole-body counting of 59Fe and direct gamma-counting of 59Fe on blood samples, this method represents a sensitive method for studying the intestinal absorption of 55Fe and 59Fe in man and at the same time allows estimation of the amount of radioiron located in the vascular compartment.

Adaptation in iron absorption: iron supplementation reduces nonheme-iron but not heme-iron absorption from food.

BACKGROUND: Results of cross-sectional studies suggest that in healthy people, iron absorption adapts to meet physiologic needs and stabilize iron stores, but this has not been adequately tested in longitudinal studies. OBJECTIVE: We tested whether heme- and nonheme-iron absorption decrease in response to increased iron intake and whether iron stores reach a steady state. DESIGN: In a randomized, placebo-controlled trial, heme- and nonheme-iron absorption by healthy men and women (n = 57) were measured before and after 12 wk of supplementation with 50 mg Fe/d as ferrous sulfate. Serum and fecal ferritin were measured during supplementation and for 6 mo thereafter. RESULTS: Initially, both heme- and nonheme-iron absorption were inversely associated with serum ferritin concentration. Volunteers who took iron supplements, even those with serum ferritin <21 microg/L (n = 5), adapted to absorb less nonheme iron (3.2% at week 12 compared with 5.0% at week 0, P: < 0.001) but not less heme iron from a beef-based meal. Serum ferritin concentration was slightly but significantly higher after iron supplementation than after placebo (difference = 13 microg/L). This higher ferritin concentration persisted for >/=6 mo after supplementation, except in subjects with low iron stores, whose serum ferritin returned to baseline within 3 mo. Fecal ferritin excretion increased 2.5-fold (P: < 0.05) during supplementation. CONCLUSIONS: Healthy individuals, even those with low iron stores, had reduced nonheme-iron absorption from food in response to iron supplementation. Despite this partial adaptation, iron stores were greater after iron supplementation than after placebo and this difference was sustained, except in individuals with the lowest iron stores.

Evaluation of uncertainties in estimates of fetal doses from 59Fe kinetic studies at Vanderbilt University.

In a 1997 paper, Stabin et al. published estimates of the fetal radiation doses for women who received oral administrations of 59Fe at Vanderbilt University in the 1940's. These authors concluded that there was "considerable uncertainty... in the amount of radioactive material administered to these subjects." In an effort to quantify this uncertainty, the underlying factors in the input data used in the Stabin et al. dose estimates have been examined in detail. Such factors include (a) an absence of detailed information on, and discrepancies in, the amounts of 59Fe reported to have been administered; (b) the probability that the radioactive iron included 55Fe as well as 59Fe; (c) uncertainties as to the period of time that elapsed between the administration of the radioiron and the taking of the maternal blood samples, and the accompanying impacts of radioactive decay; (d) possible losses of 59Fe in the procedures used in preparing the blood samples; and (e) questions as to the reported efficiency of the counting equipment. Our principal conclusion is that, due to the significant uncertainties and the lack of key information, it is not possible to estimate the doses accurately. An ancillary conclusion, however, is that the doses were probably significantly higher than previously estimated. This latter possibility should be carefully considered by any investigators who subsequently seek to use these estimates to quantify the relationship between the doses to the fetus and the resulting health effects.

Kinetic modeling of 52Fe/52mMn-citrate at the blood-brain barrier by positron emission tomography.

The kinetics of iron at the blood-brain barrier of the monkey were studied in vivo using positron emission tomography (PET) and the tracer 52Fe/52mMn-citrate. 52mMn is the beta(+)-emitting daughter nuclide of 52Fe and therefore contributes to the observed signal and background in the PET images and may influence the quantification of physiological relevant iron parameters. The kinetics of pure (52m)Mn-citrate at the blood-brain barrier of the monkey were studied experimentally, and the analysis of the data with a reasonable compartment model led to equal efflux and influx parameters for Mn (1.35 +/- 0.3 x 10(-2) min(-1)). By using complexes between Mn and diethylenetriaminepentaacetic acid, the validity of the proposed model could be confirmed. To describe the observed kinetics of 52Fe/(52m)Mn-citrate, the manganese model was coupled to an iron model, which finally allowed the quantification of two iron-specific parameters: an input rate into global brain tissue of 7.15 +/- 2.6 x 10(-4) min(-1) and a time delay of roughly 24 min to account for the observed activities. The simpler linearization procedure has been proposed and could be applied to all our data sets and is able to replace the complicated nonlinear iron/manganese tracer kinetic model neglecting any influence of manganese on the analysis.

The concept of iron bioavailability and its assessment.

In this review a broad overview of historical and current methods for the assessment of iron bioavailability was given. These methods can be divided into iron solubility studies, iron absorption studies, endpoint measures, and arithmetic models. The pros and cons of all methods were discussed. First, studies on in vitro and in vivo iron solubility have been described. The disadvantages of iron solubility include the impossibility of measuring absorption or incorporation of iron. Furthermore, only the solubility of nonheme iron, and not heme iron, can be studied. Second, we focused on iron absorption studies (either with the use of native iron, radioiron or stable iron isotopes), in which balance techniques, whole-body counting or postabsorption plasma iron measurements can be applied. In vitro determination of iron absorption using intestinal loops or cell lines, was also discussed in this part. As far as absorption studies using animals, duodenal loops, gut sacs or Caco-2 cells were concerned, the difficulty of extrapolating the results to the human situation seemed to be the major drawback. Chemical balance in man has been a good, but laborious and expensive, way to study iron absorption. Whole-body counting has the disadvantage of causing radiation exposure and it is based on a single meal. The measurement of plasma iron response did not seem to be of great value in determining nutritional iron bioavailability. The next part dealt with endpoint measures. According to the definition of iron bioavailability, these methods gave the best figure for it. In animals, the hemoglobin-repletion bioassay was most often used, whereas most studies in humans monitored the fate of radioisotopes or stable isotopes of iron in blood. Repletion bioassays using rats or other animals were of limited use because the accuracy of extrapolation to man is unknown. The use of the rat as a model for iron bioavailability seemed to be empirically based, and there were many reasons to consider the rat as an obsolete model in this respect. The double-isotope technique was probably the best predictor of iron bioavailability in humans. Disadvantages of this method are the single meal basis and the exposure to radiation (as far as radioisotopes were used). Finally, some arithmetic models were described. These models were based on data from iron bioavailability studies and could predict the bioavailability of iron from a meal.

The influence of meat on nonheme iron absorption in infants.

During weaning the infant has a high iron requirement, and highly available dietary iron is needed to ensure optimal iron status. Muscle tissue has been identified as an enhancer of nonheme iron absorption in adults, although the influence of meat on nonheme iron absorption in infants has not been previously reported. The effect of the addition of 25 g of meat (lean beef) on nonheme iron absorption from a home-prepared vegetable purée meal (80 g of vegetables) was investigated in infants in the present study. The meals did not differ in their contents of other known enhancers or inhibitors of nonheme iron absorption. Incorporation of stable isotopes of iron (57Fe and 58Fe) into red blood cells 14 d after intake was used to measure iron absorption, using a cross-over design in eight healthy infants 43-49 wk of age. Nonheme iron absorption was significantly increased (p = 0.002) from the vegetable purée with added meat (geometric mean 15.0%) compared with the puréed vegetables (geometric mean 9.9%). These results thus suggest that meat is also an enhancer of nonheme iron absorption in infants and that nonheme iron absorption from weaning foods can be increased by the addition of meat.

Improvement of the Eakins and Brown method for measuring 59Fe and 55Fe in blood and other iron-containing materials by liquid scintillation counting and sample preparation using microwave digestion and ion-exchange column purification of iron.

The simultaneous measurement of 59Fe and 55Fe in whole blood by liquid scintillation counting by the Eakins and Brown (EB) method is extensively used in iron absorption studies. The EB method requires many steps which increase the chances of error and decrease its sensitivity. We describe two modifications to the above method consisting of microwave digestion and column purification of iron. This "New Method" (NM) is simpler and more precise, and sensitive than the EB method. Counting efficiencies with the NM are similar for 59Fe (75%) as with the EB method but are better for 55Fe (29% for NM vs 22%), and cross counting from 59Fe into the 55Fe window is lower with the NM (3.7-4.5%) than with the EB method (10-12%). For the NM, recoveries of radioactive blood samples, in relation to processed standards ranged from 100 to 103% for 59Fe and 101 to 113% for 55Fe. For the EB method, recoveries ranged from 94 to 99% for 59Fe and from 88 to 93% for 55Fe. Even with very low counts, average intrarun CV with the NM was lower than 5.4% for either isotope, while it was as high as 10.0% for 55Fe with the EB method.

A turnover study in the male rat of plasma-bound 59Fe, 114Inm and 109Cd with particular reference to the gonad.

After intravenous doses of the plasma-bound radionuclides 59Fe, 114Inm and 109Cd, only a minute percentage localizes in the rat testis and remains largely unchanged with time. Intratesticular injection of appropriately reduced volumes led to much higher proportionate percentage retention of 14, 65 and 11 for 59Fe, 114Inm and 109Cd, respectively. By this route, significant feedback of the elements escaping initial binding was prevented. Distinct but different testicular turnovers were now discernible. As a receptor of fluid and spermatozoa from the testicular tubules, the epididymis provides an indication of entry into and interaction of the metals with spermatogenic cells. For 59Fe no measurable changes were detected, whereas a progressive increase in epididymal 114Inm occurred, which had not reached a plateau by 70 days. 109Cd, now demonstrated within the testicular tubules by autoradiography, remained at constant organ level for upwards of 16 days but had declined by 25% by 57 days. At this point, the epididymis showed a five-fold increase in the radionuclide, declining to one-half this value by 126 days. Since 109Cd is carrier free, the data reflect a body turnover of dietary cadmium. These results, overall, are compatible with the entry of a proportion of each radionuclide into the seminiferous tubules and reaction with spermatogenic cells. Possible interpretations of the observed differences are presented.

Duration of the inhibitory effect of calcium on non-haem iron absorption in man.

We investigated the duration of the inhibitory effect of calcium from milk and cheese (340 mg) in a breakfast meal on non-haem iron absorption from a hamburger meal eaten 2 or 4 h after the breakfast. The effect of calcium on iron absorption was studied in 21 human subjects by using paired observations and a dual-radioisotope method (55Fe and 59Fe). No duration effect of calcium on iron absorption was observed in this study. The present findings offer an opportunity for theoretical improvement of iron nutrition by a redistribution of the daily intake of calcium to the meals with a minor iron content, i.e. breakfast and the evening meal.

Erythropoiesis, serum erythropoietin, and serum IGF-I in rats during accelerated growth.

In this study we have examined the correlation between activity of erythropoiesis and serum concentrations of erythropoietin and insulin-like growth factor I in male and female rats during accelerated growth (day 30-90). We found that fractional incorporation of iron into newly formed red blood cells was linearily correlated with body weight gain. Total iron incorporation into newly formed red blood cells reflecting total daily red cell formation increased almost linearily between day 25 and 80 after birth in both sexes. While serum erythropoietin concentrations decreased in the time interval investigated (25-120 days), serum IGF-I levels increased in both sexes between day 25 and 55. In this period, individual values of total iron incorporation into red blood cells and serum IGF-I concentrations were linearily correlated. Our observations support the concept that IGF-I rather than erythropoietin modulates erythropoiesis during accelerated growth and thus manages a proportional increase in body mass and oxygen transport capacity.

Correlation between erythropoietic activity and body growth rate in hypertransfused polycythemic growing rats as the result of an erythropoietin-dependent operating mechanism.

The established relationship between erythropoietic activity and body growth rate in the polycythemic growing rat could be the result of either an erythropoietin (EPO)-dependent or an EPO-independent operating mechanism. The present study was thus undertaken to elucidate the nature of the aforementioned mechanism by assessing the ratio between plasma immunoreactive EPO (iEPO) concentration and erythropoietic activity in young hypertransfused rats for different body growth rates. Red blood cell (RBC)-59Fe uptake was about 75% in 21-day-old rats; it rapidly decreased with time when the animals were placed on a protein-free diet, approaching a level of about 1% by the 10th day of protein starvation. Over the same period plasma iEPO decreased from 55 mU/ml to 7 mU/ml. Body growth rate was 0. Following this "protein depletion period" the rats received diets containing different amounts of casein ("protein repletion period") added isocalorically to the protein-free diet to elicit a rise in body growth rate. Statistically significant relationships (p less than 0.001) were found between dietary casein concentration and body growth rate (r = 0.991), dietary casein concentration and RBC-59Fe uptake (r = 0.991), dietary casein concentration and plasma iEPO level (r = 0.992), body growth rate and RBC-59Fe (r = 0.986), and body growth rate and plasma iEPO level (r = 0.994) in hypertransfused polycythemic rats during the protein repletion period. These findings suggest that the correlation between erythropoietic activity and growth rate in the growing rat is the result of an erythropoietin-dependent operating mechanism, which appears to be independent of the ratio tissue oxygen supply/tissue oxygen demand.

Iron kinetics effects of 88 millirads. Partial-versus-total body X-irradiation.

Rats were irradiated with one tibia shielded (95% marrow exposure), total body exposed (TBI, 100%), and only one tibia exposed (5%), or they were sham irradiated (SI, 0%). Plasma Fe-59 clearance time (T1/2) and Fe-59 content ratio in the right and left tibia (RT/LT) were assayed to determine the erythroid activity of the overall marrow of the animals and the relative marrow activity in the exposed and shielded tibias, respectively. When a major fraction of the overall marrow was shielded or irradiated, the overall erythroid activity levels were identical to those of the SI and TBI animals, respectively. Interestingly, enhanced normoblastosis was observed in the marrow of the exposed tibia of individual animals exhibiting normal erythroid activity in 95% of the marrow. Conversely, localized marrow with normal erythroid activity was found in a shielded tibia of individual rats, demonstrating an enhanced erythroid activity in a major fraction of the total body. It was concluded that 88 mrad can alter marrow functions in a small isolated skeletal region as effectively as in the whole body, and tandem assays of the Fe-59 T1/2 and Fe-59 RT/LT can facilitate ultra-low-dose X-ray studies involved with partial body exposures.

[Effect of polyphenols of coffee pulp on iron absorption]. / Efecto de los polifenoles de la pulpa de café en la absorción de hierro.

The effect of the polyphenols of coffee pulp on iron absorption was studied using the method of ligated segments in rats. Optimal conditions to measure iron absorption, were determined using as criteria the concentration of Fe59 and the time that produced the highest value of blood radioactivity. A concentration of 0.4 uCi/dose of Fe59 and a 3-hr period were chosen to measure iron absorption. Experimental groups were formed assigning six rats randomly to each group. Each group was injected with a solution of 59Fe and either with the standard polyphenol solution or with the coffee pulp extract, except the control group which was injected with the Fe59 solution only. The effect of two polyphenol concentrations was also studied. Iron uptake from the duodenum was found to be the best indicator of iron absorption when compared to the sum of iron uptake by the tissues (blood, liver, spleen, kidneys, heart and carcass). Therefore, this indicator was used to interpret the results obtained. Catechin, tannic acid and the coffee pulp extract decreased significantly iron absorption when compared with the control group. The level of polyphenols used in these experiments is similar to the amounts consumed by animals fed coffee pulp at a 10% level. Therefore, we can conclude that the antinutritional effect of coffee pulp polyphenols may be partially due to their capacity to bind iron.

Efecto de los polifenoles de la pulpa de café en la absorción de hierro / Effect of polyphenols of caffee pulp on iron absorption

Con el fin de determinar el efecto de los polifenoles de la pulpa de café en la absorción del hierro, se llevó a cabo una serie de ensayos en ratas (peso promedio 200 g) utilizando el método del segmento ligado. Se estandarizaron las condiciones óptimas para medir la absorción de 59Fe usando como criterio la concentración de dicho elemento y el tiempo que produjeran mayor radiactividad en la sangre. Se escogió una concentración de 0.4 micronCi/dosis y un tiempo de tres horas para realizar los ensayos de absorción de hierro en presencia de polifenoles. Se midió la absorción de 59Fe en presencia de los polifenoles patrone (ácido tánico y catequina) y los extractos de pulpa de café en dos concentraciones, utilizando para el caso seis ratas por cada tratamiento. Se encontró que el mejor indicador de la absorción de hierro era el paso de 59Fe a través del duodeno, y no la suma de 59Fe presente en los tejidos (sangre, hígado, bazo, riñones, corazón y carcás). Por esta razón, se investigó el efecto de los polifenoles de la pulpa de café en la absorción del hierro, tomando para cada tratamiento el valor promedio de los resultados de absorción de 59Fe através del segmento ligado (100 - % 59Fe presente en el duodeno). El análisis de los resultados reveló que tanto la presencia de los polifenoles patrones, como la de los extractos de pulpa de café disminuían considerablemente la absorción de hierro al compararse con el grupo control. Los niveles de polifenoles aplicados en este estudio son comparables a los que consumen animales alimentados con dietas suplementadas con pulpa de café, aún a niveles tan bajos como 10%. Por consiguiente, es factible asegurar que el efecto antinutricional de la pulpa de café se debe en parte a la capacidad de los polifenoles de ligar el hierro de la dieta