Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.     

Blood P50 calculated from a single measurement of pH, PO2, and SO2.

P50 was calculated from a single measurement of pH, Po2, and So2 at a known temperature in 135 blood samples from 21 normal nonsmokers and eight patients. In the 92 blood samples with So2 between 20 and 90 per cent, the standard deviation of repeated calculated P50's on the same sample of blood at different So2 was plus or minus 1.0 Torr. Below So2 of 20 per cent and above So2 of 90 per cent, the standard deviations were plus or minus 5.5 and plus or minus 2.4 Torr, respectively. Combined measurement errors of plus or minus 1 Torr in Po2, plus or minus 1 percent in So2, plus or minus 0.01 in pH, and plus or minus 0.1 degree C in temperature are sufficient to explain the observed variation in calculated P50 in 90 per cent of 135 blood samples from 29 subjects and account for the greater observed variation at So2 less than 20 per cent and greater than 90 per cent.     

The influence of organic fertilizers,obtained from poplar barks,on the correction of iron chlorosis ofLupinus albus L.

Summary The influence exerted by organic matter on the correction of iron chlorosis inLupinus albus L., grown in calcareous soil, was studied through a pot experiment by incorporating two rates (1.5 per cent and 3 per cent on the soil) of three organic fertilizers obtained from poplar barks. The fertilizers differed in the length of humification process and in the integration with different materials,i.e.: H fertilizer: 8 months humified poplar barks without any addition, HF fertilizer: 4 years humified poplar barks with 4 per cent of metallic iron, HP fertilizer: 18 months humified poplar barks with 25 per cent of poultry manure. The higher rate (3 per cent) of the H fertilizer was the most efficient in reducing chlorosis symptoms and gave the greatest dry matter yield. Among the treatments with the lower rate (1.5 per cent), the HF fertilizer gave the best results; conversely, the HP fertilizer failed to correct chlorosis and to enhance yield. Therefore the experiment suggested that organic fertilizers are useful to correct iron chlorosis, provided their phosphorus content is low and they have undergone a prolonged humification process; furthermore, integration with small amounts of iron leads to a considerable saving of organic fertilizer.     

HYPERCHOLESTEROLEMIA—Use of Niacin and Niacin Combinations in Therapy

Niacin or niacin combinations were administered in dosage of 1.0 to 6.0 gm. to 31 hypercholesterolemic patients for periods up to three years. Eighty per cent were able to continue medication for long periods without significant side effects. Jaundice, apparently due to nicotinic acid, occurred in one patient. Liver toxicity will probably be a hazard in the use of this therapy.Significant and maintained serum cholesterol depression was achieved in 80 per cent of the patients who were able to take adequate dosage. Reduction in xanthomata was observed with cholesterol reduction. In some cases, when larger doses were not tolerated or were unsuccessful, combining 1.5 gm of niacin with small doses of estrogen or triparanol achieved the desired effect. Aluminum nicotinate had no important advantage over plain niacin.     

Accumulation of storage iron in patients treated for iron-deficiency anaemia.

The repletion of iron stores after treatment was studied in 38 patients with uncomplicated iron-deficiency anaemia. The serum ferritin concentration rose significantly when oral treatment was continued for two months after the attainment of a normal haemoglobin concentration. Patients treated with a total-dose infusion of iron dextran had thehighest final serum levels, which were significantly greater than in patients given Ferro-Gradumet. Oral ferrous sulphate was almost as effective as parenteral iron in producing iron stores.     

Iron Metabolism in Patients Undergoing Regular Dialysis Therapy

Thirteen patients with chronic renal failure maintained on regular renal dialysis were studied. Seven proved to have iron deficiency on the basis of marrow iron studies, reticulocyte iron uptake, and saturation of the serum iron-binding capacity. They absorbed iron when given it by mouth and were able to utilize it for haemoglobin formation. Iron-deficient patients given 600 mg of ferrous sulphate daily for three months showed an increase in haemoglobin, but the failure to replace stores of iron is probably related to their relatively limited ability to absorb iron and the variable but sometimes considerable blood loss occurring with each dialysis.The loss may be occult, and prolonged iron therapy may be required. This is most safely achieved by giving iron by mouth.     

MEASUREMENTS OF SOME CELLULAR CHANGES DURING THE FIXATION OF AMPHIBIAN ERYTHROCYTES WITH OSMIUM TETROXIDE SOLUTIONS

On average, 15 per cent of the total haemoglobin present in the blood of the newt Triturus cristatus was extracted during 45 minutes of fixation in Palade-Caulfield fixative. This extraction was reduced with fixatives buffered at pH 6.2 instead of pH 7.4. The addition of Ca⁺⁺ ions to a final concentration of 0.01 M in the fixative completely suppressed haemoglobin extraction. The effect of the pH, and the presence or absence of Ca⁺⁺ ions in the fixative, on the rate of haemoglobin extraction has been determined. During Palade-Caulfield fixation the average projected area of newt erythrocytes increased by 37 per cent, and after dehydration and embedding in Epon the average area was 25 per cent greater than that of the unfixed cell. Fixatives buffered at pH 6.2 and containing 0.01 M Ca⁺⁺ ions caused cellular shrinkage, with the average projected area decreasing by 10 per cent in the fixative. This shrinkage continued during dehydration, and the final average area of the erythrocytes in Epon was 26 per cent less than that of the unfixed cells. Similar measurements with erythrocytes of Amphiuma tridactylum showed that after Palade-Caulfield fixation the average cellular area was increased by 45 per cent, and after dehydration and embedding in Araldite it was 36 per cent greater than that of the unfixed cell. The average nuclear area increased by 35 per cent during fixation but after embedding it was 26 per cent greater than that of the unfixed nuclei. With a fixative at pH 6.2 containing 0.01 M Ca⁺⁺ ions, both the nucleus and the whole cell shrank during fixation. The nuclear area decreased by 20 per cent and the cellular area by 22 per cent. After dehydration and embedding in Araldite, the average nuclear area had decreased by 35 per cent and the cellular area by 40 per cent. It has been shown that OsO₄ fixation lowers the isoelectric points of haemoglobins and other proteins. This finding has been used in the interpretation of the observed cellular changes resulting from fixation.     

Disparity between dietary iron intake and iron status of children aged 10-12 years

Iron status was assessed in a representative sample of 188 adolescents living in a medium-sized city in Poland. Dietary intakes were evaluated using records of diet over a period of seven consecutive days. Subjects were considered to be iron deficient when two or more of the following parameters were abnormal: serum ferritin, transferrin saturation or mean corpuscular haemoglobin concentration. Based on this definition, the prevalence of iron deficiency in the investigated sample of children aged from ten to twelve years was 12.7%. Iron deficiency anaemia was defined using the following criteria: haemoglobin values less than 12.0 g. dl (-1) in girls or less than 12.2 g. dl(-1) in boys, combined with an iron deficiency. With such a definition, the prevalence of iron deficiency anaemia in all subjects was 6.3%. Four boys (3.9%) and six girls (6.8%) were diagnosed as anaemic. The values for Hb in the anaemic boys ranged from 10.9 to 12.2 g. dl (-1) and in anaemic girls from 8.7 to 12.0 g. (-1). It was found that the majority of the individuals studied had a dietary haem-iron intake lower than that recommended. No relationship was found between the level of serum ferritin and total iron and vitamin C dietary intake, but there was positive correlation between serum ferritin and intake of haem iron. A seven-day dietary history questionnaire correctly identified children at risk of iron deficiency anaemia.     

Current status in iron chelation in hemoglobinopathies

Although blood transfusions are important for patients with hemoglobinopathies, chronic transfusions inevitably lead to iron overload as humans cannot actively remove excess iron. The cumulative effects of iron overload lead to significant morbidity and mortality, if untreated. Desferrioxamine (DFO) is the reference-standard iron chelator whose safety and efficacy profile has been established through many years of clinical use. DFO side effects are acceptable and manageable however the prolonged subcutaneous infusion regimen of 5-7 days per week is very demanding and results in poor adherence to therapy. Deferiprone (Ferriprox, L1) is a bidentate molecule, orally administrable three-times/day, licensed in Europe and in other regions but in the USA and Canada, for the treatment of iron overload in patients for whom DFO therapy is contraindicated or inadequate. Preliminary evidences suggest that Deferiprone may be more effective than DFO in chelating cardiac iron. The side effects include gastrointestinal symptoms, liver dysfunction, joint pain, neutropenia and agranulocytosis. A weekly assessment of white blood cell counts is recommended because of the risk of agranulocytosis. Deferasirox is a new, convenient, once-daily oral iron chelator that has demonstrated in various clinical trials good efficacy and acceptable safety profile in adult and pediatric patients affected by transfusion-dependent thalassemia major and by different chronic anemias (SCD, BDA, MDS). The long half-life of Deferasirox (16-18 hours) provides sustained 24 hr iron chelation coverage. The efficacy and safety profile have been evaluated in more than 1000 patients in clinical trials allowing FDA registration. Patient satisfaction with Deferasirox was superior than with DFO therapy.     

Causes of Early Childhood Deaths in Urban Dhaka,Bangladesh

Data on causes of early childhood death from low-income urban areas are limited. The nationally representative Bangladesh Demographic and Health Survey 2007 estimates 65 children died per 1,000 live births. We investigated rates and causes of under-five deaths in an urban community near two large pediatric hospitals in Dhaka, Bangladesh and evaluated the impact of different recall periods. We conducted a survey in 2006 for 6971 households and a follow up survey in 2007 among eligible remaining households or replacement households. The initial survey collected information for all children under five years old who died in the previous year; the follow up survey on child deaths in the preceding five years. We compared mortality rates based on 1-year recall to the 4 years preceding the most recent 1 year. The initial survey identified 58 deaths among children <5 years in the preceding year. The follow up survey identified a mean 53 deaths per year in the preceding five years (SD±7.3). Under-five mortality rate was 34 and neonatal mortality was 15 per thousand live births during 2006–2007. The leading cause of under-five death was respiratory infections (22%). The mortality rates among children under 4 years old for the two time periods (most recent 1-year recall and the 4 years preceding the most recent 1 year) were similar (36 versus 32). The child mortality in urban Dhaka was substantially lower than the national rate. Mortality rates were not affected by recall periods between 1 and 5 years.     

Effect of 60Co gamma-irradiation on the reaction of mixed disulphides of mercaptoethylguanidine with enzymes of rat-liver cytoplasm.

A theoretical assumption of Eldjarn and Pihl suggests that mixed disulphides formed by radioprotective aminothiols and protein SH-groups can be broken down by enzymes in the organism, and the native structure of the macromolecules restored. Irradiation should enhance this effect. In our experiments, mixed disulphides of mercaptoethylguanidine (MEG) and albumin/haemoglobin were split by the soluble enzyme fraction of rat-liver homogenate (cytosol). The liberation of the radioprotector MEG is brought about by small molecules; dialysed cytosol has no effect, nor has the suspension of particles of mitochondria. On irradiation with doses in the 0-1--5-0 Mrad range, the mixed disulphide bridge is stabilized and made more resistant to splitting. Increased resistance up to 700 per cent with albumin-MEG and 160 per cent with haemoglobin (Hb)-MEG mixed disulphide was observed compared with the unirradiated control.     

45Ca displacement related to pharmacologically induced prolonged action potentials in Nitella flexilis

Nitella cells were loaded with 45Ca2+ to an activity of 2 X 10(5) cpm. Insertion of two glass-capillary electrodes into each of six cells released varying amounts of Ca2+ in the order of 1 mumol per cell, but hyperpolarizing and depolarizing pulses up to 500 ms in duration caused no measurable loss (less than 57 pmol) of Ca2+ even when the latter elicited action potentials. Addition of 10 mumol of Ba2+ or tetraethylammonium (TEA) caused losses up to 1200 pmol of Ca2+ from the cells and prolonged the action potentials by a factor of three or more. Subsequent addition of Ba2+ or TEA to treated cells caused no further losses of Ca. Because prolonged action potentials can apparently only be elicited after the chelation or displacement of Ca2+, we propose that, as in many animal cells, the K+ channels responsible for the normal brief repolarizing phase of the action potential are controlled by Ca2+ in these electrically excitable plant cells.     

ISIS-2: 10 year survival among patients with suspected acute myocardial infarction in randomised comparison of intravenous streptokinase,oral aspirin,both, or neither

Objective: To assess effects of intravenous streptokinase, one month of oral aspirin, or both, on long term survival after suspected acute myocardial infarction. Design: Randomised, “2×2 factorial,” placebo controlled trial. Setting: 417 hospitals in 16 countries. Subjects: 17 187 patients with suspected acute myocardial infarction randomised between March 1985 and December 1987. Follow up of vital status complete to at least 1 January 1990 for 95% of all patients and to mid-1997 for the 6213 patients in United Kingdom. Interventions: Intravenous streptokinase (1.5 MU in 1 hour) and oral aspirin (162 mg daily for 1 month) versus matching placebos. Main outcome measures: Mortality from all causes during up to 10 years’ follow up, with subgroup analyses based on 4 year follow up. Results: After randomisation, 1841 deaths were recorded in days 0-35, 991 from day 36 to end of year 1, 1478 in years 2-4, and 1230 in years 5-10. Allocation to streptokinase was associated with 29 (95% confidence interval 20 to 38) fewer deaths per 1000 patients during days 0-35. This early benefit persisted (death rate ratio 0.98 (0.92 to 1.04) for additional deaths between day 36 and end of year 10), so that there were 28 (14 to 42) and 23 (2 to 44) fewer deaths per 1000 patients treated with streptokinase after 4 years and 10 years respectively. There was no evidence that absolute survival benefit increased with prolonged follow up among any category of patient, including those presenting early after symptoms started or with anterior ST elevation. Nor did the early benefits seem to be lost in any category (including those aged over 70). Allocation to one month of aspirin was associated with 26 (16 to 35) fewer deaths per 1000 during first 35 days, with little further benefit or loss during subsequent years (death rate ratio 0.99 (0.93 to 1.06) between day 36 and end of year 10). The early benefit obtained with combination of streptokinase and one month of aspirin also seemed to persist long term. Conclusions: The early survival advantages produced by fibrinolytic therapy and one month of aspirin started in acute myocardial infarction seem to be maintained for at least 10 years.

Key messages

• Large randomised trials have shown that the survival benefits of intravenous fibrinolytic therapy for patients with acute myocardial infarction persist for at least one year, but there is relatively little information about longer term effects
• By contrast, this report from the ISIS-2 trial of intravenous streptokinase and of one month of oral aspirin includes nearly 4000 deaths between the start of year 2 and the end of year 10
• The early survival benefits of fibrinolytic therapy persist for at least 10 years after treatment and do not seem to increase or decrease with prolonged follow up in any category of patients, including elderly subjects
• The survival benefits of short term aspirin treatment in acute myocardial infarction also persist long term and are additional to those of fibrinolytic therapy, and other studies show that these benefits can be increased by continuing aspirin treatment for some years after myocardial infarction

     

A quantitative study on recovery from acute haemorrhage in adult laboratory rats. I. Blood morphology

We evaluated changes in blood morphology for detecting acute blood loss in laboratory rats. Haematologic indices which are normally used in investigating drug effects on rat blood morphology were examined. Sublethal acute blood loss (25 ml per kg body weight) modified rat blood morphology in a few minutes; the blood picture was normal within two weeks. Blood drawing (usually up to 10 ml per kg b.w.) can modify rat blood morphology within one week. Erythrocyte counts, haemoglobin concentration and haematocrit decreased earliest, reticulocyte counts were changed longest. Comparing our results and literature data the recovery of blood morphology from acute haemorrhage seems to be more rapid in rats than in men.     

Liver iron depletion and toxicity of the iron chelator Deferiprone (L, CP20) in the guinea pig

The use of the iron chelator deferiprone (L, CP20, 1,2-dimethyl-3-hydroxypyrid-4-one) for the treatment of diseases of iron overload and other disorders is problematic and requires further evaluation. In this study the efficacy, toxicity and mechanism of action of orally administered L were investigated in the guinea pig using the carbonyl iron model of iron overload. In an acute trial, depletion of liver non-heme iron in drug-treated guinea pigs (normal iron status) was maximal (approximately 50% of control) after a single oral dose of L1 of 200 mg kg, suggesting a limited chelatable pool in normal tissue. There was no apparent toxicity up to 600 mg kg. In each of two sub-acute trials, normal and iron-loaded animals were fed L (300 mg kg day) or placebo for six days. Final mortalities were 12/20 (L) and 0/20 (placebo). Symptoms included weakness, weight loss and eye discharge. Iron-loaded as well as normal guinea pigs were affected, indicating that at this drug level iron loading was not protective. In a chronic trial guinea pigs received L (50 mg kg day) or placebo for six days per week over eight months. Liver non-heme iron was reduced in animals iron-loaded prior to the trial. The increase in a wave latency (electroretinogram), the foci of hepatic, myocardial and musculo-skeletal necrosis, and the decrease in white blood cells in the drug-treated/normal diet group even at the low dose of 50 mg kg day suggests that L may be unsuitable for the treatment of diseases which do not involve Fe overload. However, the low level of pathology in animals treated with iron prior to the trial suggests that even a small degree of iron overload (two-fold after eight months) is protective at this drug level. We conclude that the relationship between drug dose and iron status is critical in avoiding toxicity and must be monitored rigorously as cellular iron is depleted.     

Alpha 1-antitrypsin deficiency in childhood

Alpha-1-antitrypsin is a blood glycoprotein synthesized in the liver. It is a protease inhibitor of the serpine group and has a specific action for elastase. Alpha-1-antitrypsin electrophoresis shows about 20 phenotypes, the normal one being PiM. The allele PiZ is usually responsible for liver or lung disease in children or adults, respectively. Eleven per cent of PiZZ infants present with prolonged neonatal cholestasis. Twenty-five of 45 PiZZ infants with prolonged neonatal cholestasis presented with later cirrhosis. Persistence of jaundice beyond the sixth month of age, early development of splenomegaly, persistence of hard hepatomegaly and liver function abnormalities, and early portal fibrosis have a poor prognostic significance. The most severe course occurs in infants with an early histologic pattern of paucity of interlobular bile ducts. Portal hypertension was present in 19 of 25 children presenting with cirrhosis; 8 of 25 PiZZ children with cirrhosis died during childhood.     

THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : I. CONDITIONS CONTROLLING ABSORPTION AND RETENTION OF POTASSIUM.

1. Individual variations in the potassium content of the fresh muscles of frogs are notable even when computed as percentages of the dry solids. The potassium content averaged higher in freshly collected summer frogs than in winter frogs after a period of captivity. 2. Muscles show a loss of from 8 to 15 per cent of their potassium during perfusion with potassium-free Ringer solution but tenaciously hold the remainder. 3. Muscles, stimulated to contract under conditions that do not produce irreversible stages of fatigue, show losses of potassium no greater than those attributable to the presence of a potassium-free medium. 4. A condition favorable to the taking up of potassium probably occurs in a contracting muscle because rubidium and cesium, substances very similar to potassium in chemical and physiological behavior, are absorbed in retainable form by a contracting muscle but not by a resting one.     

A simple and rapid method for the determination of "free" iron in biological fluids

We present a convenient method for determining "free" or non-protein-bound iron in biological fluids. The new method is based on the bathophenantroline method for determination of total serum iron, and comprises binding of iron by a chromogenic chelator (bathophenantroline-disulphonate, BPS), which is specific for ferrous iron. The ferrous complex of BPS absorbs strongly at 535 nm, and the detection limit is less than 1 μM in a sample size of 50 μl. The chelator does not liberate iron from either haemoglobin or transferrin. Interference from copper or zinc in concentrations up to 50 μM does not significantly disturb measurements. The main problem when measuring in blood plasma, the high and fluctuating background in the region around 535 nm, has been overcome through filtering techniques. Data from measurements of ferrous iron in microdialysate, cerebrospinal fluid, and blood plasma in different animal models and clinical conditions are presented as illustrative examples of the usefulness of the method. The method allows the determination of ferric, as well as ferrous, iron in the same sample.     

Bioavailability of heme iron in biscuit filling using piglets as an animal model for humans

The objective of this work was to evaluate the bioavailability of heme iron added to biscuit filling. It comprised two stages: first, the development of the heme iron enriched biscuit filling; second, the evaluation of the bioavailability of the mineral in fattening piglets. Two groups were selected randomly and fed: a) Low iron feed and biscuits with heme iron supplemented filling; b) Normal feed (with ferrous sulphate). Weight and blood parameters were measured every fifteen days. Averages were compared after duplicate analyses. The filling had a creamy appearance, chocolate taste and smell, appropriate spreadability, heme iron content of 2.6 mg per gram and a shelf-life of a month. The heme iron supplemented pigs registered a greater (P<0.05) weight gain (27.8% more than the control group). Mortality in the heme iron group was 10%, compared to 50% in the control group. The amount of iron measured in the different compartment was greater in the heme group (3315 mg) than in the control group (2792 mg). However, the amount of iron consumed in the latter was greater. We show that an acceptable product with high heme iron content can be formulated, suitable for use as biscuit filling. The heme iron supplement produced better weight increase and lesser mortality in fattening pigs. The bioavailability of heme iron was 23% greater (P<0.05) compared to ferrous sulphate.     

Cost benefits of low dose subcutaneous erythropoietin in patients with anaemia of end stage renal disease.

OBJECTIVE--To assess the cost benefits of low dose subcutaneous recombinant human erythropoietin in correcting the anaemia of end stage renal disease. DESIGN--Three year retrospective study. SETTING--Subregional nephrology service serving a mixed urban and rural population of 800,000. SUBJECTS--60 patients with symptoms of anaemic end stage renal disease treated with erythropoietin (43 receiving haemodialysis; 11 receiving continuous ambulatory peritoneal dialysis; two with predialysis end stage renal disease; four with failing renal transplants). MAIN OUTCOME MEASURES--Costs and savings of achieving and maintaining a haemoglobin concentration of 85-105 g/l with erythropoietin. RESULTS--All patients treated with erythropoietin achieved the target haemoglobin concentration at median induction doses of 97 (95% confidence interval 95 to 108) units/kg/week, and this was maintained with 79 (75 to 95) units/kg/week at an average annual cost per patient of 2260 pounds. Admissions related to anaemia were virtually eliminated (246 v 1 inpatient days for 12 months before and after starting erythropoietin). 54 patients required no blood transfusions after starting erythropoietin, and the total requirements fell from 230 to 21 units in the 12 months before and after starting erythropoietin. Iron stores were maintained with oral or intravenous iron. All patients reported increased wellbeing, appetite, and exercise capacity. Hypertension developed or worsened in 30 patients, resulting in hospital admissions in five patients, one of whom had seizures. CONCLUSION--Low dose subcutaneous erythropoietin restores haemoglobin concentrations sufficiently to abolish blood transfusion requirements and reduce morbidity. The net cost of erythropoietin prescribed in this way (2260 pounds/patient/year) was largely offset by savings in costs of hospital admissions. The true annual cost to the NHS was around 1200 pounds per patient.