Duodenal iron proteins in idiopathic hemochromatosis.

This study was undertaken to assess the relationship between iron absorption and the concentration of duodenal iron proteins in normal subjects and patients with idiopathic hemochromatosis (IH). Biopsies were obtained endoscopically from the duodenum in 17 normal subjects, 3 of whom were mildly iron deficient, and 7 patients with untreated IH. The absorption of both heme and nonheme iron was increased in IH despite a 20-fold elevation in serum ferritin. Immunoassays using MAb were used to measure transferrin, H-rich ferritin, and L-rich ferritin in mucosal samples. Mucosal transferrin concentrations in normal subjects did not correlate with either iron status or iron absorption, indicating that mucosal transferrin plays no physiological role in iron absorption. Mucosal transferrin was significantly lower in IH, presumably because of a decrease in mucosal transferrin receptors. Mucosal H and L ferritin concentrations were directly related to body iron stores and inversely related to iron absorption in normal subjects. In IH, mucosal H and L ferritin failed to increase in parallel with the serum ferritin, but were appropriate for the level of iron absorption. The relationship of mucosal H/L ferritin in IH did not differ from that observed in normal subjects. Our findings indicate that the major abnormality in duodenal iron proteins in IH is a parallel decrease in the concentration of H- and L-rich ferritin. It is not evident whether this is the result or the cause of the absorptive abnormality.

In vitro growth modulation by L-ascorbic acid of colony-forming cells from bone marrow of patients with myelodysplastic syndromes.

In vitro colony growth was studied on bone marrow cells from 51 patients with myelodysplastic syndromes (MDS), using a cell culture method with the unique feature of daily feeding, in an effort to gain insight into the pathophysiology of MDS and to assess the clinical utility of this cell culture assay. The colony growth pattern of MDS marrow cells is remarkably similar to that of acute myeloid leukemia but quite dissimilar from that of normal marrow, in support of a common pathophysiological mechanism for these two disorders. In particular, L-ascorbic acid (LAA) enhanced colony growth in 30% and suppressed growth in 16% of cases, a finding also similar to that in acute myeloid leukemia, indicating a unique growth requirement which may be explored for therapeutic purposes. Further, these LAA effects have prognostic value, with LAA-sensitive (both LAA-enhanced and LAA-suppressed) cases displaying shorter survivals than LAA-insensitive cases (median survival of 5 months versus 18 months; P = 0.011). This prognostic value is independent of, and more powerful than, bone marrow blasts; the median survival was 18 months for less than 5% bone marrow blasts and 8 months for greater than 5% bone marrow blasts (P = 0.044). These two risk factors can be used together to identify patients with an extremely good or an extremely poor prognosis. This study establishes the clinical usefulness of the LAA effect in MDS as a prognostic factor and provides a new lead to explore in understanding differential biochemical/molecular events and, possibly, a new therapeutic approach to the management of MDS.

Structure of a eukaryotic decoding region A-site RNA.

The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects.

Structural origins of aminoglycoside specificity for prokaryotic ribosomes.

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.

Myelosuppression in Legionnaires' disease.

A patient undergoing examination for moderately severe renal insufficiency had fulminant five-lobed pneumonia caused by Legionella pneumophila. Her clinical course was complicated by granulocytopenia. Bone marrow aspiration showed notable inhibition of myelopoiesis, involving all stages of maturation. The presence of a serum inhibitor of granulopoiesis was demonstrated by in vitro bone marrow culture. Normal bone marrow cultured in the presence of serum from two normal control subjects yielded 69 +/- 5.2 (mean +/- SE) and 61 +/- 5 granulocyte colonies. When the patient's serum was substituted for normal human serum only 14.3 +/- 2.3 colonies were formed. Each of the drugs to which she had been exposed was tested in the in vitro system, using therapeutic concentrations, and none showed significant suppression of granulocyte colony formation. These observations indicate that legionnaires' disease was associated with a serum factor that notably inhibited the growth of granulocyte colonies in this patient.

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.

Assessment of the role of nonheme-iron availability in iron balance.

To assess the nutritional relevance of absorption studies that use extrinsically labeled single meals, we developed a method for measuring nonheme-iron absorption from the diet and compared the results with absorption from single meals. When subjects consumed their usual diet, there was good agreement between dietary absorption (6.4%) and representative single meals fed in the laboratory (6.1%). Nonheme-iron availability, as estimated by a model that incorporated the effect of both enhancers and inhibitors, correlated significantly with absorption from single meals but not with dietary absorption. When the diet was modified to promote iron absorption maximally, dietary absorption increased only slightly (8.0%) and remained significantly lower than it was from single meals (13.5%). With an inhibitory diet, the decrease in absorption from single meals was similarly exaggerated. These results indicate that in the context of a varied Western diet, nonheme-iron bioavailability is less important than absorption studies with single meals would suggest.

Inhibition of food iron absorption by coffee.

Dual isotope studies were performed in iron replete human subjects to evaluate the effect of coffee on nonheme iron absorption. A cup of coffee reduced iron absorption from a hamburger meal by 39% as compared to a 64% decrease with tea, which is known to be a potent inhibitor of iron absorption. When a cup of drip coffee or instant coffee was ingested with a meal composed of semipurified ingredients, absorption was reduced from 5.88% to 1.64 and 0.97%, respectively, and when the strength of the instant coffee was doubled, percentage iron absorption fell to 0.53%. No decrease in iron absorption occurred when coffee was consumed 1 h before a meal, but the same degree of inhibition as with simultaneous ingestion was seen when coffee was taken 1 h later. In tests containing no food items, iron absorption from NaFeEDTA was diminished to the same extent as that from ferric chloride when each was added to a cup of coffee. These studies demonstrate that coffee inhibits iron absorption in a concentration-dependent fashion.

Reduction of the soy-induced inhibition of nonheme iron absorption.

Recent studies in normal men have demonstrated that soy products have a pronounced inhibitory effect on the absorption of nonheme dietary iron. The purpose of the present investigation was to explore approaches to overcoming this inhibition. A modest but significant increase in iron absorption was observed when test meals containing either isolated soy protein or whole soybeans were baked at 200 degrees C. The addition of 100 mg ascorbic acid to a meal containing isolated soy protein had a more dramatic effect, increasing mean absorption from 0.6 to 3.2%. A similar but less pronounced increase was observed when meat was added to the isolated soy protein meal. These results indicate that the inhibitory effect of soy can be reversed to some extent by substances known to enhance nonheme iron absorption.

The inhibitory effect of soy products on nonheme iron absorption in man.

Radioiron absorption studies were performed in male volunteer subjects to determine the effect on nonheme iron absorption of various semipurified proteins. When egg albumen and casein were substituted in protein-equivalent quantities in a semisynthetic meal, similar mean absorptions of 2.5 and 2.7% were observed. In contrast, isolated soy protein reduced absorption sharply, to an average of 0.5%. When egg albumen in the semisynthetic meal was replaced with full fat soy flour, textured soy flour, and isolated soy protein, absorption fell from 5.5 to 1.0, 1.9, and 0.4%, respectively, indicating an inhibitory effect by a wide range of soy products. The effect of substituting textured soy flour for meat in a meal containing a hamburger, french fries, and a milkshake was also evaluated. With 3:1 and 2.1 ratios of meat to unhydrated textured soy flour, absorption decreased by 61 and 53%, respectively. The soy products tested in this study have a pronounced inhibitory effect on the absorption of nonheme iron.

Inhibitory effect of a soybean-protein--related moiety on iron absorption in humans.

The inhibitory effect of soybean protein isolates on nonheme-iron absorption was studied in 34 human subjects. Iron absorption was measured by using an extrinsic radioiron label in liquid-formula meals containing hydrolyzed corn starch, corn oil, and either egg white or a series of soybean-protein derivatives. The unmodified soybean-protein isolate markedly inhibited iron absorption. Percentage absorption was 19-fold higher when an extensively enzyme-hydrolyzed preparation with very little phytate was used as the protein source. Both the glycinin (11S) and conglycinin (7S) fractions of soybean protein were inhibitory to iron absorption. Dephytinization removed the inhibitory effect of the glycinin but not of the conglycinin fraction. We conclude that there are two major inhibitors of iron absorption in soybean-protein isolates, phytic acid and a protein-related moiety contained in the conglycinin (7S) fraction.

Iron absorption in humans: bovine serum albumin compared with beef muscle and egg white.

We studied the influence of bovine serum albumin and beef meat on nonheme iron absorption in humans and on dialyzable iron in vitro. The addition of serum albumin to a maize gruel had no significant effect on nonheme Fe absorption whereas the addition of beef meat caused a threefold increase. When added to a bread meal, serum albumin caused a modest 60% increase in nonheme Fe absorption and beef meat had no effect. When added to a protein-free meal, serum albumin reduced Fe absorption by 47% compared with a 72% reduction on addition of egg white. The bioavailability of nonheme Fe from meals containing serum albumin was consistently overestimated by the in vitro technique. We conclude that the facilitation of nonheme Fe absorption by meat is not a general property of all animal protein but is better explained by the action of one or more specific animal tissues.

Iron status of elderly Americans.

Studies of iron nutriture in the elderly are limited and very few include observations on individuals over the age of 75. The two Health and Nutrition Examination Surveys carried out by the United States Department of Health, Education and Welfare demonstrate that the mean iron intake of Americans is adequate until the age of 75. However, with changes in the major food sources there is a decrease in iron derived from meat and a concomitant rise in the proportion supplied by breakfast cereals. Alterations in dietary iron bioavailability++ that may result from this have not been studied. Physiological data suggest that the elderly do not represent a target population for iron deficiency since iron requirements are no greater than those of adult men and lower than those of children and menstruating women. Furthermore, there is little direct evidence of a high prevalence of iron deficiency in the elderly, but the laboratory measurements that have proved useful in defining iron status in younger people have not been standardized for or extensively used in older people. Anemia is still the most important known consequence of significant iron deficiency. However, the application of Hb or hematocrit standards used in younger people to the elderly as well as the assumption that anemia can be equated with iron deficiency invalidates the conclusions of many surveys. Hb and hematocrit measurements are not suitable screening tests for iron deficiency in the elderly and there is an urgent need for a clearer understanding of the physiological and nutritional factors responsible for lower Hb values in older people, particularly older Blacks.

Iron availability from infant food supplements.

Radioiron absorption tests are performed in human volunteer subjects to measure iron absorption from five infant food supplements. These products included corn-soya-milk, corn-soy blend, wheat-soy blend, wheat protein concentrate blend, and whey-soy drink mix. In iron replete adult males, mean percentage absorption ranged from 0.6 to 1.4%. By relating these results to absorption from a reference dose of inorganic iron, it was estimated that iron-deficient infants would absorb between 1.7 and 4.1% of the iron contained in the infant foods. These results indicate that this fortification iron is poorly absorbed, and probably supplies only about half of the daily iron requirement for iron deficient children between 6 months and 3 yr of age.

Iron fortification of infant cereals: a proposal for the use of ferrous fumarate or ferrous succinate.

Hemoglobin-repletion tests in rats, organoleptic studies, and iron-absorption studies in humans were used to search for Fe sources with high bioavailability that could be added to infant cereals as alternatives to the Fe compounds currently used for fortification. From rat and organoleptic studies on 11 alternative Fe sources, ferrous fumarate, ferrous succinate, and ferric saccharate were selected as the most suitable for infant-cereal fortification and, by use of radioactive labels, absorption of those compounds from fortified cereal was measured in adult human volunteers. There was no difference in absorption between ferrous fumarate and ferrous sulfate whereas the values for ferrous succinate, ferrous saccharate (10% Fe), and ferric pyrophosphate were 92%, 74%, and 39% of the ferrous sulfate values, respectively. We conclude that ferrous fumarate and ferrous succinate are highly available Fe sources in man that can be used to fortify infant cereals without causing fat oxidation or discoloration.

Iron absorption in humans as influenced by bovine milk proteins.

The effect of the two major bovine milk protein fractions on the dialyzability of iron in vitro under simulated gastrointestinal conditions and on the absorption of Fe by humans was studied. Liquid-formula meals were prepared from hydrolyzed maize starch, corn oil, and either spray-dried egg white or a milk-protein product. In meals containing egg white, 3.32% of the Fe was dialyzable. The substitution of casein and whey protein products reduced the dialyzable fraction to 0.19-0.56% and 0.86-1.60%, respectively. Percentage Fe absorption was also reduced by the substitution of casein or whey protein for egg white. Mean absorption values fell from 6.67 to 3.65% and 2.53 to 0.98%, respectively. When the intact milk-protein products were replaced by enzyme- or acid-hydrolyzed preparations, the dialyzable fraction increased markedly and in proportion to the extent of hydrolysis. A similar but much smaller effect on absorption was observed. These studies suggest that bovine casein and whey proteins are responsible at least in part for the poor bioavailability of the Fe in some infant formulas.

Iron absorption from legumes in humans.

Iron bioavailability in a variety of legumes was determined by radioisotopic measurements of iron absorption in human subjects. Soybeans, black beans, lentils, mung beans, and split peas were prepared as soups, labeled by the extrinsic tag method, and served to fasting subjects. Mean percentage absorption was uniformly low, ranging from 0.84 to 1.91%. Differences in absorption between the means were not statistically significant. The results indicate that these five commonly eaten legumes are all poor sources of dietary iron.

Soy protein products and heme iron absorption in humans.

Dual radioiron tags were used to measure both heme and nonheme iron absorption simultaneously from meat-containing meals in 76 healthy male volunteers. Partial substitution of beef with soy flour reduced the availability of nonheme iron but improved the percentage absorption of heme iron significantly (27 to 59% rise). In contradistinction three other powerful inhibitors of nonheme iron absorption, bran, tea, and desferrioxamine, had no appreciable effect on heme. Ascorbic acid (100 mg and 1000 mg in separate experiments) improved nonheme iron uptake markedly but also failed to alter the assimilation of heme. These studies demonstrate that the deleterious effects on iron nutrition of substituting soy protein for beef are partially offset by improved availability of the remaining heme iron as well as by an increase in the nonheme iron content of the meal.

Soy protein, phytate, and iron absorption in humans.

The effect of reducing the phytate in soy-protein isolates on nonheme-iron absorption was examined in 32 human subjects. Iron absorption was measured by using an extrinsic radioiron label in liquid-formula meals containing hydrolyzed corn starch, corn oil, and either egg white or one of a series of soy-protein isolates with different phytate contents. Iron absorption increased four- to fivefold when phytic acid was reduced from its native amount of 4.9-8.4 to less than 0.01 mg/g of isolate. Even relatively small quantities of residual phytate were strongly inhibitory and phytic acid had to be reduced to less than 0.3 mg/g of isolate (corresponding to less than 10 mg phytic acid/meal) before a meaningful increase in iron absorption was observed. However, even after removal of virtually all the phytic acid, iron absorption from the soy-protein meal was still only half that of the egg white control. It is concluded that phytic acid is a major inhibitory factor of iron absorption in soy-protein isolates but that other factors contribute to the poor bioavailability of iron from these products.

Interaction of iron with other nutrients.

Iron is essential for oxygen transport, oxidative metabolism, and cellular growth. Interactions between iron and other dietary factors play a significant role in determining the adequacy of iron nutrition and have important implications for food fortification in developing countries. Vitamin A and vitamin C deficiency states may affect iron transport, metabolism, and storage within the body.