Glycosylation of hair: possible measure of chronic hyperglycaemia.

To determine whether hair is excessively glycosylated in diabetes mellitus 4 cm hair samples were taken proximally from behind the ear in 50 white non-diabetics and 46 diabetics. Hair glycosylation was assayed by a modification of the thiobarbituric acid reaction. Blood was taken from the diabetics at the same time for measurement of glycosylated haemoglobin concentration. The mean (1 SD) concentration of fructosamine (mumol/100 mg hair) was 0.054 (0.011) for normal hair. Glycosylation was not related to sex, age, or hair colour. The diabetics'' hair was more heavily glycosylated (0.097 (0.045] than normal (p less than 0.01) and there was a correlation between hair glycosylation and the concentration of glycosylated haemoglobin in the diabetics (r = 0.71; p less than 0.01). Hair from non-diabetics showed a stable time related increase in glycosylation when incubated with glucose. Glycosylation of hair might provide a stable long term measure of tissue glycosylation, useful in the investigation of microvascular complications of diabetes mellitus.     

Effect of betacarotene on protein glycosylation in alloxan induced diabetic rats

Free radicals are increasingly formed in diabetes mellitus by the auto oxidation of glucose and glycosylated proteins. Oxidative stress and proteinglycosylation are closely related processes and have been shown to contribute to the development of complications in diabetes mellitus. The extent of protein glycosylation was assessed in alloxan induced diabetic rats after being treated with 50 mg of betacarotene for 40 days. The level of fructosamine and glycosylated haemoglobin was comparison with non treated diabetic rats. The results indicate the beneficial role of betacarotene in reducing diabetic complications like glycosylation in experimental diabetic rats.     

Entamoeba histolytica secretes two haem-binding proteins to scavenge haem

Entamoeba histolytica is a human pathogen which can grow using different sources of iron such as free iron, lactoferrin, transferrin, ferritin or haemoglobin. In the present study, we found that E. histolytica was also capable of supporting its growth in the presence of haem as the sole iron supply. In addition, when trophozoites were maintained in cultures supplemented with haemoglobin as the only iron source, the haem was released and thus it was introduced into cells. Interestingly, the Ehhmbp26 and Ehhmbp45 proteins could be related to the mechanism of iron acquisition in this protozoan, since they were secreted to the medium under iron-starvation conditions, and presented higher binding affinity for haem than for haemoglobin. In addition, both proteins were unable to bind free iron or transferrin in the presence of haem. Taken together, our results suggest that Ehhmbp26 and Ehhmbp45 could function as haemophores, secreted by this parasite to facilitate the scavenging of haem from the host environment during the infective process.     

Red cell distribution width and erythrocyte osmotic stability in type 2 diabetes mellitus

This study aimed to investigate the relationship between red cell distribution width (RDW) and erythrocyte osmotic stability in non-diabetic and diabetic individuals in both sexes. The study sample (N = 122) was constituted by 53 type 2 diabetics (DM) and 69 non-diabetics (ND), being 21 and 22 men in each group, respectively. The osmotic stability of erythrocytes was obtained by the variation in saline concentration (dX) capable of determining hypoosmotic lysis. Higher RDW values and lower serum iron concentrations were found in the diabetic group when compared to the non-diabetic volunteers. In the group of diabetic women, RDW was positively correlated with the reticulocyte index, and both RDW and dX were negatively correlated with iron, haemoglobin, transferrin saturation index, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. In all the groups studied, RDW was positively correlated with dX, especially in the diabetic group, where the correlation was the strongest. RDW elevation in both women and men with type 2 diabetes mellitus was associated with decreased serum iron indicators. Furthermore, RDW has a similar meaning to dX, as small erythrocytes have less haemoglobin, resulting in both an increase of RDW and dX.     

Estimates of normal binding of a human recombinant alpha interferon to peripheral blood mononuclear cells from a study matching healthy subjects to subjects with insulin dependent diabetes

This study compares the binding of a human recombinant alpha-interferon to peripheral blood mononuclear cells (PBMC) from patients with insulin dependent diabetes (IDDM) mellitus and control subjects. Diurnal and longer term of variation, feeding, fasting and haemoglobin glycosylation were examinated for their influence on interferon binding to PBMC. No gross differences in binding were demonstrated, in particular no effect of glucose levels was seen on the binding of interferon alpha-2 to PBMC.     

Chemical kinetics measurements on the reaction between blood and ozone

The pseudofirst-order ozonization rate constant of whole bovine blood has been measured in comparison to that of free haemin. The free prosthetic group haemin (which has also the central iron atom in the oxidized form) shows k values in the range of 0.20-0.03 s(-1) while the haeme groups inside haemoglobin protein and contained in the whole blood sample show slightly lower k values, just in the range of 0.10-0.02 s(-1). It has been found that ozone even with whole blood reacts specifically with haemoglobin of the red cells because it is adsorbed selectively on the iron atoms of the haeme prosthetic groups of haemoglobin. The absorption implies the oxidation of the central iron atom of the haeme groups with formation of methaemoglobin followed by an oxidative fission of the haeme rings. The other blood components do not exert any significant protection to the reaction between ozone and haemoglobin, which appear extremely specific and selective like the reaction between CO or HCN and haemoglobin. By analogy with the behaviour of these other gases ozone may be classified as a blood poison. The results of this work are discussed in the frame of the risks connected to the ozonotherapy and autohaemotherapy involving the blood ozonization of human or animal subjects and the re-injection of ozonized blood into the bodies.     

Iron Increases Diabetes-Induced Kidney Injury and Oxidative Stress in Rats

Diabetic nephropathy is both a common and a severe complication of diabetes mellitus. Iron is an essential trace element. However, excess iron is toxic, playing a role in the pathogenesis of diabetic nephropathy. The present study aimed to determine the extent of the interaction between iron and type 2 diabetes in the kidney. Male rats were randomly assigned into four groups: control, iron (300-mg/kg iron dextran), diabetes (a single dose of intraperitoneal streptozotocin), and iron + diabetes group. Iron supplementation resulted in a higher liver iron content, and diabetic rats showed higher serum glucose compared with control rats, which confirmed the model as iron overload and diabetic. It was found that iron + diabetes group showed a greater degree of kidney pathological changes, a remarkable reduction in body weight, and a significant increase in relative kidney weight and iron accumulation in rat kidneys compared with iron or diabetes group. Moreover, malondialdehyde values in the kidney were higher in iron + diabetes group than in iron or diabetes group, sulfhydryl concentration and glutathione peroxidase activity were decreased by the diabetes and iron + diabetes groups, and protein oxidation and nitration levels were higher in the kidney of iron + diabetes group as compared to iron or diabetes group. However, iron supplementation did not elevate the glucose level of a diabetic further. These results suggested that iron increased the diabetic renal injury probably through increased oxidative/nitrative stress and reduced antioxidant capacity instead of promoting a rise in blood sugar levels; iron might be a potential cofactor of diabetic nephropathy, and strict control of iron would be important under diabetic state.     

Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.     

Energy expenditure in children with type I diabetes: evidence for increased thermogenesis.

The aim of the study was to assess whether increased energy expenditure causes the negative energy balance (tissue catabolism) commonly seen in children with insulin dependent (type I) diabetes. Resting metabolic rate and thermogenesis induced by adrenaline were measured in five healthy children and 14 children with type I diabetes who were all free of clinical signs of late complications of diabetes mellitus but differed in their degree of glycaemic control (in eight glycated haemoglobin concentration was less than 10% and in the six others greater than or equal to 10%). When compared with the control subjects children with type I diabetes had normal resting metabolic rates but their urinary nitrogen excretion was significantly raised (11.5 (SD 5.4) mg/min in those with glycated haemoglobin concentration less than 10%, 11.6 (5.2) mg/min in those with concentration greater than or equal to 10% v 5.4 (3.0) mg/min in control subjects). During the infusion of adrenaline the diabetic children showed a threefold and sustained increase in thermogenesis and disproportionate increases in the work done by the heart, in lipid oxidation rate, and in plasma concentrations of glucose, free fatty acids, and ketone bodies. The increased thermogenic effect of adrenaline did not correlate with the degree of glycaemic control. Increased thermogenesis may explain the tissue wasting commonly seen in children with type I diabetes during intercurrent stress.     

Distribution of non-enzymatically bound glucose in in vivo and in vitro glycosylated type I collagen molecules

Non-enzymatic glycosylation of collagen occurs both in vivo during diabetes and in vitro after incubation with glucose. Glycosylated collagen exhibits altered physicochemical and biological properties which could explain some of the complications of diabetes. To provide a mechanistic explanation of this modification the localization of bound glucose was investigated using NaB[3H]H4 reduction and CNBr cleavage. Glucose fixation is distributed mainly on the alpha 1CB6 peptide after in vitro glycosylation whereas this distribution occurs less specifically during diabetes. It is concluded that fibrillogenesis alteration of in vitro glycosylated collagen is related to glucose fixation on free epsilon NH2 sites normally implied in intermolecular interactions.     

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.     

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

Haemoglobin biosynthesis site in rabbit embryo erythroid cells

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Effect of Iron on Erythropoietin Production in Anaemic Piglets

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

Bacterial iron homeostasis

Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FeoB. For pathogens, host-iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by down-regulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels.     

Effects of waterborne iron on growth, reproduction, survival and haemoglobin in Daphnia magna

The effects of waterborne iron (FeCl3 X 6H2O) on growth, reproduction, survival and haemoglobin content in Daphnia magna were studied from subnormal to toxic concentrations in hard reconstituted water. Low concentrations of iron stimulated reproduction and haemoglobin synthesis after chronic exposure for 21 days. Maximum reproduction occurred between 0.1 and 1 microgram Fe 1(-1). Juvenile growth was not stimulated by iron but was slightly inhibited between 1 and 8 micrograms Fe 1(-1) and above 128 micrograms Fe 1(-1). A slight inhibition of growth persisted for 21 days. Total haemoglobin content was above the control with no waterborne iron at all but one concentration (512 micrograms Fe 1(-1]. The highest value (3.8 X control value) was found at 2 micrograms Fe 1(-1). The haemoglobin content decreased between 64 and 512 micrograms Fe 1(-1) and increased at higher concentrations. The decrease coincided with an inhibited reproduction. The increase was found in non reproductive survivors. A comparison with a previous study in D. magna suggests that ambient conditions (hardness and pH) and ageing of the water are important for the effects of waterborne iron. At a hardness of 250 mg 1(-1) as CaCO3 and a pH range of 7.0-8.0 the ZEP (Zero Equivalent Point) for reproduction was 158 micrograms Fe 1(-1). Continuous exposure to higher concentrations is expected to lead to extinction of a D. magna population.     

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.