Transferrin: internal homology in the amino acid sequence.

Two regions of the primary structure of human serum transferrin, of 87 and 57 residues, are reported. When these are suitably aligned by placing two gaps, 40 percent of the amino acids in corresponding positions are identical. This indicates that the doubling of an ancestral structural gene occurred during the evolution of the transferrins.

Quantitative analysis of pregnancy-associated plasma proteins in human placenta.

By immunochemical methods and simultaneous measurements of several normal plasma proteins, human placenta was shown to contain elevated quantities of four pregnancy-associated plasma proteins (PAPP's). In the order of increasing amounts, PAPP-A, PAPP-C, PAPP-B, and human chorionic somatomammotropin (PAPP-D) all were present in placenta extracts in quantities greater than could be expected on the basis of their content in maternal blood. In sharp contrast, the placental content of pregnancy zone protein could be entirely accounted for by the maternal plasma present in the placenta. All of the PAPP's appeared to be readily extractible from placental tissue with buffered saline, the large bulk of them being solubilized in the first extraction procedure. However, absorption studies indicated that appreciable quantities of the PAPP's were still present in the insoluble placental residue after 12 sequential extractions with saline. The chorioamniotic membranes were not significantly enriched in any of the PAPP's. Immunochemical analysis of unwashed placental tissue extracts for the PAPP's IgA, and IgM (maternal blood derived), as well as albumin and transferrin (maternal and fetal blood derived), permitted calculations to be made of the amount of blood and PAPP's in placenta. On the basis of these data, it was roughly estimated that a 400-g placenta (wet weight) would occupy 312 ml in volume, and would contain 144 ml of blood. Of this blood, 36 ml would be derived from the mother.

Comparison of electron paramagnetic resonance and atomic absorption serum copper measurements in human normal control and cancer patients.

Serum copper levels of 132 controls and 122 cancer patients (including Hodgkin's disease, lung cancer, breast cancer, leukemia, untreated patients, and patients in progression) were measured using both atomic absorption (AA) and electron paramagnetic resonance (EPR) techniques. The data pairs were compared using linear regression analysis, EPR versus AA, and all the data (controls and cancers) fit a single regression line with a squared correlation coefficient (r2) of 0.80. Comparison of the subpopulations revealed possible small differences, but none great enough to be of diagnostic value for individual patients. Thus, in a number of cases of practical interest, EPR-determined serum copper levels were essentially redundant with respect to AA measurements. Previous reports recommending the use and possible superiority of EPR for serum copper determinations appear not to have compared the EPR and AA techniques adequately or correctly. EPR serum copper measurements may yet provide unique data in these diseases, but a more detailed analysis of the spectral parameters will be required. EPR-determined serum transferrin levels are also reported.

Chelator-mediated iron efflux from reticulocytes.

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.

Transferrin-binding and iron-binding proteins of rabbit reticulocyte plasma membranes. Three distinct moieties.

1. Transferrin-membrane complexes and iron-binding membrane complexes were solubilized with sodium dodecyl sulfate from the plasma membranes of reticulocytes that had been incubated with (59Fe,125I)-labeled transferrin. Gel filtration of solubilized material demonstrated 125I-labeled transferrin complexed to two moieties, a minor component (Peak I) of apparent molecular weight 435,000 and a major component (Peak II) of apparent molecular weight 200,000. Most of the membrane 59Fe was located in Peak I. 2. Sepharose-bound anti-transferrin was used to purify the 125I-labeled transferrin-membrane complexes. The 59Fe/125I ratio in the transferrin complex purified from Peak I was the same as in the original transferrin and thus contained membrane-bound transferrin to which the 59Fe was still attached. The 59Fe/125I ratio in the purified Peak II transferrin complex was 0.33 times that of the original transferrin, indicating that more than 60% of its 59Fe had been delivered to the reticulocyte. 3. The purified transferrin complexes analyzed by SDS-polyacrylamide gel electrophoresis demonstrated a single band of apparent molecular weight 78,000 both by Coomassie blue stain for protein and by 125I radioactivity. The specific activity of this material was 0.27 and 0.56 times that of the original transferrin for Peak I and Peak II, respectively, indicating that transferrin in Peak I and II was bound to a membrane component with a molecular weight similar to that of transferrin. 4. The isoelectric focusing pattern of the Peak II transferrin complex showed isoelectric points of pH 6.7 and 6.2 compared to pH 5.4 for transferrin. 5. On the basis of these studies we propose that transferrin is first bound to a membrane protein and then delivers iron to a membrane component distinct and separate from the transferrin-binding moiety. Prior to its release, transferrin markedly depleted of iron is still bound to a component in the plasma membrane.

Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding.

Endocytosis and intracellular processing of transferrin (Tf) and Tf receptors were examined in rat reticulocytes. Subcellular fractionation revealed that Tf enters a non-lysosomal endocytic compartment with a density between those of plasma membrane and lysosomes. After 20 min of uptake at (37 degrees C) 35 to 40% of cell-associated Tf was contained in this intermediate-density compartment. To test the fidelity of colloidal gold-Tf (AuTf) as a probe for Tf processing, reticulocytes were fractionated after uptake of 131I-Tf and 125I-AuTf. The subcellular distributions of the two ligands were indistinguishable by this method, a result suggesting that AuTf is processed similarly to Tf. Electron microscopy revealed that AuTf entered multivesicular endosomes (MVEs) as well as various small vesicles and tubular structures. In addition MVE exocytosis was observed with discharge of inclusion vesicles and associated AuTf. AuTf was bound to the outside of these vesicles both before and after exocytosis. These data suggest that Tf receptors are shed from developing reticulocytes by incorporation into the limiting membrane of inclusion vesicles, followed by discharge of these vesicles by MVE exocytosis. As further evidence of this process, we isolated inclusion vesicles after their discharge and found them to contain Tf receptors. Moreover, the rate of Tf receptor shedding by inclusion vesicle discharge matches Tf receptor loss rates closely enough to suggest that this is the primary path of receptor loss during reticulocyte development.

An assay for erythropoietin in vitro at the milliunit level.

A method is described for the assay of erythropoietin using primary cultures of adult rat bone marrow cells. Either total labeled iron uptake by the cells or hematin synthesis from labeled iron may be used as the measure of erythropoietin action. The method is useful in the range 0.001 to 0.010 U, where the log response is linear with the log dose, and can be carried out in 2 working days. This method has the disadvantage of detecting asialoerythropoietin which has no activity in vivo.

Familial hemochromatosis: characteristics of the precirrhotic stage in a large kindred.

Ffty asymptomatic members of a kindred with familial hemochromatosis were studied in an effort to clarify some of the physiologic abnormalities present in the pre-cirrhotic or latent stage of the disease. Using excess hepatic iron as a marker for inheritance of hemochromatosis, results of liver biopsies on 31 family members suggest an auto-somal dominant mode of inheritance with incomplete expressivity. In addition to a relationship between alcohol intake and excess liver iron, there was a strong association between the level of alcohol intake and the presence of hepatic fibrosis in those subjects with excess iron stores. Both serum iron and transferrin saturation were significantly higher in family members with iron overload than in those who were not affected. Only transferrin saturation was significantly correlated with the severity of hepatic iron deposition. Studies of glucose tolerance (OGTT, IVITT, glucose clamp studies) demonstrated a defect in carbohydrate metabolism associated with deficient insulin secretion and insulin resistance, both of which were related to the degree of hepatic iron depostion. In this kindred we have found no evidence for a contribution of inheritance to the carbohydrate intolerance of hemochromatosis. Iron overload was not related to activity of hepatic collagen proline hydroxylase or urinary excretion of peptide-bound hydroxyproline. Serum ferritin, previously thought to be a reliable marker of reticuloendothelial iron stores, was normal in 19 of 20 family members with iron overload.

Screening test for iron overload.

It is presently estimated that 2-3/10(3) people living in the United States are homozygous for the iron-loading gene and are thereby susceptible to severe iron overload. The complications of this disorder can be prevented if recognized in the asymptomatic precirrhotic stage. We have developed a simple, inexpensive screening method for its early laboratory detection based on the fact that one of the earliest laboratory manifestations of hemochromatosis is a reduction in unsaturated iron-binding capacity. The sequential addition of a small measured quantity of saturating iron and bathophenanthroline disulfonate will result in color with highly saturated but not with normal sera. This screening test almost completely differentiated between 40 normal individuals and 40 patients with iron overload (as defined by a transferrin saturation greater than 55%). The test provides a cost-effective method for screening large segments of the population to detect this genetic disorder.

Predictive ability of various nutritional variables for mortality in elderly people.

Nutritional indices (percentage ideal body weight [IBW], serum albumin, serum transferrin, total lymphocyte count [TLC] and delayed cutaneous hypersensitivity [DH] response) were assessed in 80 consecutive patients (aged 85-100 y) within 24 h of admission to determine their predictive value for mortality. Nine patients died. Pearson correlation analysis demonstrated that death was significantly (p less than 0.05 to less than 0.01) associated with sepsis, serum albumin less than 30 g/L, TLC less than or equal to 1500 cells/mm3, and percentage IBW less than or equal to 90%. However, when serum albumin was controlled for, logit regression analyses demonstrated that the impact of other nutritional indices on death was insignificant. The effect of serum albumin remained significant (p less than 0.05 to less than 0.01) even when age and physician's diagnosis were held constant. With the logit model, serum albumin greater than or equal to 30 g/L had a sensitivity of 0.33, specificity of 0.99, and overall predictive power of 0.91. Serum albumin is thus the simplest and best single predictor of mortality and can provide early identification of elderly people at increased risk of death.

The intake and excretion of lead and cadmium by the elderly.

Metabolic balance for lead and cadmium were carried out in 23 healthy elderly people aged 69.7 to 85.5 yr while living in their own homes and eating self-selected diets. Mean intakes of lead and cadmium were 54.6 and 8.6 micrograms/day, with mean retentions of -8.7 and -1.7 micrograms/day, respectively. Daily dietary lead correlated (p less than 0.05) with the intake of energy, nitrogen, calcium, iron, and zinc but not with manganese or copper. Dietary intake of cadmium correlated (p less than 0.05) only with that of zinc and manganese. There was a highly significant (p less than 0.001) inverse correlation between the percentage cadmium absorbed and body iron stores measured as serum iron, percentage iron saturation, and ferritin. Mean whole blood concentrations were 138 micrograms/l for lead and 0.79 microgram/l for cadmium. The negative balances observed in these elderly people were very different from the positive balances found in a previous similar study in children.

The iron status of Black and white female adolescents from eight Southern states.

Hb, hematocrit, plasma iron, and transferrin saturation were measured in approximately 1000 girls aged 12, 14, or 16 yr in eight southern states. The iron status parameters did not differ significantly among the three age groupings or between menstruating and nonmenstruating girls. Blacks had significantly lower mean Hb (p less than 0.0001), hematocrit (p less than 0.0001), and transferrin saturation (p less than 0.05) levels than whites and a greater proportion of Blacks exhibited low Hb (p less than 0.05) and low hematocrit levels (p less than 0.01). Adjusting for dietary iron intakes and per capita income levels did not adequately account for significant race differences for iron status parameters. These findings support the contention that genetic as well as environmental factors are responsible for the frequently reported Black-white differences in Hb and hematocrit levels.

Detection of protein-calorie malnutrition in the elderly.

The geriatric population is an ever-increasing segment of our society. A careful review of the physical, anatomic, and biochemical changes which occur with aging indicates that measurements traditionally made to detect the presence of protein-calorie malnutrition may be inappropriate for the elderly due to a lack of specific norms for comparison. Changes that are normally seen with age may appear to be representative of a nutritional deficit if the observer is unfamiliar with the aging process. Methods of extrapolating existing nutritional assessment data for use in the geriatric population, development of norms specific for the aged, and study of nontraditional methods of evaluation should all be considered in an effort to improve nutritional assessment techniques for this population.

Transferrin: assay of myotrophic effects and method for immunocytochemical localization.

1. Primary cultures of dissociated embryonic chicken skeletal muscle cells provide an ideal model for investigating the effects of growth factors such as Tf because these cells undergo a highly integrated pattern of differentiation and maturation. 2. The trophic effects of a growth factor such as Tf can be assessed on muscle cultures by the determination of such parameters as acetylcholinesterase and acetylcholine receptors. These proteins are specific to the cultured myotubes, appear in high levels following fusion of myoblasts into myotubes, and are relatively easy to assay. 3. Tf and other growth factors are internalized by a receptor-mediated mechanism (see Trowbridge et al. and Seligman and Allen, this volume). These growth factors can be localized to specific tissues by immunocytochemistry at the light or electron microscopic level. This information on cellular distribution could be very useful in assessing the pattern of growth and differentiation with regard to the particular growth factor under study.

Interaction of iron deficiency and lead and the hematologic findings in children with severe lead poisoning.

Microcytic anemia, long considered an effect of lead poisoning, may in fact result from coexisting iron deficiency. In this study, how RBC size, hemoglobin, and zinc protoporphyrin vary as a function of iron status in a group of children with high lead levels was examined. Charts of all children (N = 51) admitted to Cook County Hospital for treatment of lead poisoning in 1981 to 1983 were reviewed for data on age, blood lead level, hemoglobin concentration, MCV, transferrin saturation and zinc protoporphyrin level. The mean lead level was 86 micrograms/dL and the range was 63 to 190 micrograms/dL. Children with transferrin saturation values less than 7% had a mean MCV of 56 microL, hemoglobin of 8.9 g/dL, and zinc protoporphyrin of 693 micrograms/dL; for those with saturations of 7% to 16%, the values were 61 microL, 10.1 g/dL, and 581 micrograms/dL, respectively; the children with saturations greater than 16% had normal mean MCVs and hemoglobin concentrations (74 microL and 11.4 g/dL) and a mean zinc protoporphyrin value of 240 micrograms/dL (P less than .0005). Multiple linear regression was used to correct for effect of age, and transferrin saturation remained the most important predictor of MCV, hemoglobin, and zinc protoporphyrin levels; the addition of lead did not improve the models. Results of this study suggest that iron deficiency is strongly associated with some of the observed toxicities of lead. Also, lead poisoning can exist without producing microcytosis or anemia, and zinc protoporphyrin concentration may not be a sensitive indicator of lead level in the absence of iron deficiency.

The absorption of iron as supplements in infant cereal and infant formulas.

The absorption of iron was measured from isotopically tagged salts used in supplementing infant cereals and as the iron supplement in cow's milk and soy-based formulas. Iron as sodium iron pryophosphate and ferric orthophosphate were poorly absorbed from infant cereal (mean, smaller than 1.0%) and thus are not dependable sources of iron to meet the nutritional needs of infants. Reduced iron of very small particle size and ferrous sulfate when added to cereal was absorbed to a greater extent (mean, 4.0% and 2.7% respectively). For technical reasons, these two forms of iron had not been added to commercial cereal products because of discoloration, distribution problems of the iron in the product, and shortened shelf life. Therefore, at the present time, iron supplementation of infant cereals with sodium iron pyrophosphate, ferric orthophosphate, and reduced iron of large particle size does not provide a predictable and available source of iron to meet the needs of infants. Supplemental iron as ferrous sulfate in milk- and soy-based formulas gave a mean absorption of 3.4% and 5.4%. The iron supplements in these formulas can essentially meet the needs for dietary iron of healthy infants.

Macular degeneration and elevated serum ceruloplasmin.

Macular degeneration associated with age and drusen, an important cause of visual loss, is associated clinically with alterations in the retinal pigmented epithelium. Because the pigmented epithelium is a copper-rich tissue with antioxidant properties, the copper economy in patients and controls were studied by measuring ceruloplasmin. Ceruloplasmin, a multifunctional, copper-binding alpha-globulin, was significantly elevated in non-related patients as compared with controls (691 +/- 153 mg/L vs 312 +/- 64; P less than .001), both by the p-phenylenediamine oxidation technique and radial immunodiffusion assay. When 53 members of a large family were divided clinically into persons with and without macular degeneration, the ceruloplasmin concentrations were not significantly different from each other, but were elevated as compared with non-related controls (P less than .001). These differences were not due to an intragroup age mismatch. A group of patients with retinitis pigmentosa had normal serum ceruloplasmin concentrations. This study suggests a relationship between serum ceruloplasmin, trace metals, and the tissue alterations associated with macular degeneration that deserves further investigation.

The effect of collection technique on tear composition.

Selected proteins were quantitated after collecting samples of the tears by using two sampling techniques. Tears from the same individual were collected via absorption by Schirmer filter paper strip from the unanesthetized, inferior, conjunctival sac and were compared with tears collected by a capillary tube (taking care not to touch the conjunctiva), after stimulation of tearing by irritation of the nasal mucosa with ammonia vapor. Tear samples were quantitated immunochemically for two typical lacrimal proteins, lysozyme and lactoferrin, and three typical serum proteins, albumin, transferrin, and IgG. Tear analysis of all constituents were performed on a single sample of tears collected by each method from the same individual. Normal subjects without ocular pain or discomfort comprised a sample of 12 subjects ranging in age from 19 to 57 years and consisting of 9 men and 3 women. Concentrations of lysozyme and lactoferrin in samples collected by either method were not significantly different. In contrast, the concentration of albumin, IgG and transferrin collected by Schirmer filter paper technique was significantly higher (P less than 0.01) than the concentration in tears collected by the capillary tube technique. A highly significant increase in serum proteins was seen when the Schirmer filter paper strip was used to collect tears compared to tears collected without mechanical stimulation of the conjunctiva.

Effect of iron deficiency on tissue oxygen delivery in cyanotic congenital heart disease.

To test the hypothesis that tissue oxygen delivery would be affected by diminished oxygen stores in cyanotic congenital heart disease, serum ferritin, transferrin saturation, hemoglobin, red cell mean corpuscular volume (MCV), red cell 2,3-diphosphoglycerate (DPG), P50, blood gases, oxygen saturations and systemic oxygen transport were measured in 29 hypoxemic infants and children. For the group, aortic saturation was 81 +/- 9%, PaO2 was 50 +/- 12 mm Hg, hemoglobin 16.2 +/- 2.1 gm/dl and systemic oxygen transport 620 +/- 145 ml/min/m2. P50 was increased above normal values (28.8 +/- 2.3 vs 26.6 +/- 1.1 mm Hg, p less than 0.01), and DPG was 2.35 +/- 0.54 mumol/ml, at the upper limits of normal for this assay. Iron deficiency was present in 8. When patients with P50 greater than or equal to 30 mm Hg and P50 less than 30 mm Hg were compared, iron stores were diminished in the high P50 group: [serum ferritin (19 +/- 8 vs 53 +/- 48 ng/ml, p = 0.0006), transferrin saturation (11 +/- 6 vs 23 +/- 11%, p = 0.003) and MCV (79 +/- 8 vs 86 +/- 4 fl, p = 0.05)]. Hemoglobin, aortic oxygen saturation, PaO2 and systemic oxygen transport were similar in both groups. In children with iron sufficiency, 15 of 21 had MCV greater than 90th percentile for age and sex (p less than 0.001 versus expected distribution). Also, MCV greater than 90th percentile for age and sex had a positive predictive value of 0.88 for iron sufficiency. This study demonstrates that diminished iron stores in cyanotic congenital heart disease are associated with a more right-shifted oxyhemoglobin dissociation curve (increased P50).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of iron stores in anemic geriatric patients.

Of patients referred to a geriatric service, 66 were identified who were clearly anemic (hemoglobin less than 12 g in men, less than 11 g in women) but whose cause of anemia was not readily identifiable by noninvasive measures. The difficulty in distinguishing iron deficiency from chronic disease as a cause of anemia by noninvasive means (serum iron, total iron binding capacity, transferrin saturation ratio, and serum ferritin), is highlighted by the poor power of these investigations when compared with bone marrow iron stores. A transferrin saturation ratio of less than 11% and a serum ferritin of less than 45 pg/L serve better than currently accepted values to identify iron deficiency in this population.