Effect of iron supplementation on iron status during the first week after blood donation.

INTRODUCTION: Frequent blood donations may lead to a negative iron balance. Iron depletion may be prevented by iron supplementation after whole blood donations. The aim of this study was to compare the short time changes in iron status after donation in two groups randomized to iron supplementation or no additional iron. A second objective was to evaluate the effect of iron supplementation in donors having HFE-variants compared to HFE wild types. METHODS: Subjects of both genders (199 women, 200 men) were randomised to receive iron supplementation or no additional iron after donation. Iron status, defined by the concentration of haemoglobin, serum ferritin, soluble transferrin receptor, concentration of haemoglobin in reticulocytes (CHr) and percent hypochrome mature red blood cells, was determined at the start of donation and 8 +/- 2 days after donation. HFE genotyping was performed at reappearance. RESULTS: There was a significant difference between the two study groups on all the iron status parameters. CHr was an efficient, early marker of ongoing synthesis of haemoglobin. Heterozygosity for the HFE variants C282Y and H63D had no statistically significant influence on the iron status. The donor's baseline serum ferritin value may be basis for an individual iron supplementation regimen, as donors with serum ferritin >50 microg/l do not seem to utilize the iron supplementation, but prefer endogenous iron to restore the loss of haemoglobin. CONCLUSION: Iron supplementation had a significant positive impact on the restoration of iron status one week after donation.

Iron status in Norwegian blood donors: comparison of iron status in new blood donors registered in 1993-1997 and in 2005-2006.

BACKGROUND AND OBJECTIVES: The impact of a poor iron status on the difficulties to keep recruitment of new donors at pace with the ongoing increased demand for blood transfusions was studied by comparing the iron status of new donors recruited in 1993-1997 and in 2005-2006. MATERIALS AND METHODS: Iron status was defined by haemoglobin and serum ferritin. Inclusion criteria for approving new donors were haemoglobin >/= 12.5 g/dl for women and >/= 13.5 g/dl for men, and serum ferritin > 15 microg/l for both genders. Data were gathered retrospectively from 943 subjects (55% women) in the 1990 ties and prospectively from 1013 subjects (63% women) 10 years later. RESULTS: In women, there was a significant fall in haemoglobin and serum ferritin mean values from 13.2 to 13.1 g/dl and from 30.9 to 26.9 microg/l, respectively. Rejection due to low haemoglobin was significantly increased from 14% to 24%. In men, there were minor changes that did not affect rejection rates. CONCLUSION: Iron status of women who want to serve as blood donors has deteriorated in the last 10 years, leading to an increased rejection due to haemoglobin below the inclusion criterion for blood donors.

Tetradecylthioacetic acid attenuates dyslipidaemia in male patients with type 2 diabetes mellitus, possibly by dual PPAR-alpha/delta activation and increased mitochondrial fatty acid oxidation.

AIM: We previously demonstrated that a modified fatty acid, tetradecylthioacetic acid (TTA), improves transport and utilization of lipids and increases mitochondrial fatty acid oxidation in animal and cell studies. We conducted an exploratory study of safety and effects of this novel drug in patients with type 2 diabetes mellitus and investigated the mechanism of action in human cell lines. METHODS: Sixteen male patients with type 2 diabetes mellitus received 1 g TTA daily for 28 days in an open-labelled study, with measurement of parameters of lipid metabolism, glucose metabolism and safety (ClinicalTrials.gov NCT00605787). The mechanism of action was further investigated in a human liver cell line (HepG2) and in cultured human skeletal muscle cells (myotubes). RESULTS: Mean LDL cholesterol level declined from 4.2 to 3.7 mmol/l (p < 0.001), accompanied by increased levels of the HDL apolipoproteins A1 and A2, and a decline in LDL/HDL ratio from 4.00 to 3.66 (p = 0.008). Total fatty acid levels declined, especially the fraction of the polyunsaturated n-3 fatty acids docosahexaenoic acid (-13%, p = 0.002) and eicosapentaenoic acid (-10%, p = 0.07). Glucose metabolism was not altered and the drug was well tolerated. In cultured liver cells, TTA acted as a pan-PPAR agonist with predominant PPAR-alpha and PPAR-delta activation at low TTA concentrations. In myotubes, TTA and a PPAR-delta agonist, but not the PPAR-alpha or PPAR-gamma agonists, increased the fatty acid oxidation. CONCLUSIONS: We demonstrate for the first time that TTA attenuates dyslipidaemia in patients with type 2 diabetes mellitus. These effects may occur through mechanisms involving PPAR-alpha and PPAR-delta activation, resulting in increased mitochondrial fatty acid oxidation.

The effect of blood donation frequency on iron status.

INTRODUCTION: The effects of blood donation on iron status in donors without iron supplementation were studied. Analysing interactions between donations and iron status markers may predict these effects. MATERIALS AND METHODS: Haemoglobin (Hb) and serum ferritin were analysed in 893 donors over 1 year. Serum transferrin receptor (sTfR) was measured at the first and last donation. RESULTS: Prolonged intervals prevented decrease in Hb in women and in ferritin for both genders. In women, a high TfR-F index (sTfR/log ferritin) predicted fall in Hb. CONCLUSION: Adjusting the donation intervals is a way to prevent iron deficiency in blood donors.

Prevalence of iron deficiency and anemia among healthy women of reproductive age in Bhaktapur, Nepal.

OBJECTIVE: To determine the prevalence of anemia and iron status as assessed by biochemical markers and to explore the associations between markers of iron status and iron intake. STUDY AREA AND POPULATION: Five hundred healthy women of reproductive age from the Bhaktapur district of Nepal were included in the study. METHODS: A cluster sampling procedure was applied for this cross-sectional study. Women without any ongoing infection aged 13-35 years were selected randomly from the population. We measured the plasma concentration of hemoglobin (Hb), ferritin and transferrin receptors. Dietary information was obtained by a food frequency questionnaire and two 24-h dietary recalls. RESULTS: The prevalence of anemia (Hb concentration <12 g/dl) was 12% (n=58). The prevalence of depleted iron stores (plasma ferritin <15 microg/l) was 20% (n=98) whereas the prevalence of iron deficiency anemia (anemia, depleted iron stores with elevated transferrin receptor i.e. >1.54 mg/l) was 6% (n=30). Seven percent (n=35) of women were having iron-deficient erythropoiesis (depleted iron stores and elevated transferrin receptor but normal Hb). Out of the 58 anemic women, 41 (71%) and 31 (53%) were also having elevated plasma transferrin receptor and depleted iron stores, respectively. Fifty-four percent of the women ate less than the recommended average intake of iron. The main foods contributing to dietary iron were rice, wheat flour and green and dry vegetables. CONCLUSIONS: The prevalence of anemia in our study was substantially lower than the national figure for non-pregnant women. Only about half of the women with anemia were also having depleted iron stores, suggesting that other causes of anemia may be prevalent in this population. SPONSORSHIP: Norwegian Universities Committee for Development, Research and Education (NUFU).

Proteomic-based biomarker discovery with emphasis on cerebrospinal fluid and multiple sclerosis.

Discovery of disease specific biomarkers in human body fluids has become an important challenge in clinical proteomics. Facing the increasing threat of degenerative and disabling diseases like cancer, cardiovascular, neurological and inflammatory diseases in large parts of the world's population, there is an urgent need to improve early diagnostics. In this review we discuss possibilities and limitations connected to using mass spectrometry based proteomics in the search for novel biomarkers, with focus on multiple sclerosis as a typical representative for the large group of non-curable degenerative and disabling disease with the lack of specific tests for early diagnosis. Careful control of the pre-analytical phase including sampling, storage and fractionation of samples, in addition to a thoroughly considered patient selection, is important in order to avoid false biomarkers to appear in the resulting mass spectra. Furthermore, advanced computational tools are needed in order to discover potential biomarkers from the enormous data amounts generated by the mass spectrometers. The development of such computer tools is a research field currently in the start phase and could prove to be a bottle neck in the biomarker discovery the next years. Therefore, a rather detailed review of the most used computational and pre-analytical methods is given in this review. Mass spectrometry based biomarker discovery is undoubtedly still in its early infancy. However, in light of the potential of this technology to provide deep coverage of the body fluid proteomes, it will certainly consolidate its role in developing molecular medicine into clinical practice.

The prevalence of anemia and iron deficiency is more common in breastfed infants than their mothers in Bhaktapur, Nepal.

BACKGROUND/OBJECTIVES: Iron deficiency anemia is a widespread public health problem, particularly in low- and middle-income countries. Maternal iron status around and during pregnancy may influence infant iron status. We examined multiple biomarkers to determine the prevalence of iron deficiency and anemia among breastfed infants and explored its relationship with maternal and infant characteristics in Bhaktapur, Nepal. SUBJECTS/METHODS: In a cross-sectional survey, we randomly selected 500 mother-infant pairs from Bhaktapur municipality. Blood was analyzed for hemoglobin, ferritin, total iron-binding capacity, transferrin receptors and C-reactive protein. RESULTS: The altitude-adjusted prevalence of anemia was 49% among infants 2-6-month-old (hemaglobin (Hb) <10.8 g/dl) and 72% among infants 7-12-month-old (Hb <11.3 g/dl). Iron deficiency anemia, defined as anemia and serum ferritin <20 or <12 µg/l, affected 9 and 26% of infants of these same age groups. Twenty percent of mothers had anemia (Hb <12.3 g/dl), but only one-fifth was explained by depletion of iron stores. Significant predictors of infant iron status and anemia were infant age, sex and duration of exclusive breastfeeding and maternal ferritin concentrations. CONCLUSIONS: Our findings suggest that iron supplementation in pregnancy is likely to have resulted in a low prevalence of postpartum anemia. The higher prevalence of anemia and iron deficiency among breastfed infants compared with their mothers suggests calls for intervention targeting newborns and infants.

Relevance of ferritin-binding sites on isolated mitochondria to the mobilization of iron from ferritin.

Iron can be released from ferritin and utilized by isolated rat liver mitochondria for the synthesis of heme. Mobilization of iron from ferritin is initiated by the binding of ferritin to the mitochondria in an manner compatible with binding sites or receptors for ferritin on the mitochondria. The binding completes rapidly, it is independent of temperature, saturable, reversible and enhanced by K+ and Mg2+. The amount of ferritin binding sites is approx. 0.8 pmol/mg mitochondrial protein, and the affinity constant is 6.4 . 10(6)M-1. The binding kinetics correlate well with the functional features of the ferritin-mitochondrial interaction: i.e. mobilization of iron from ferritin followed by insertion of the iron into heme. The results support the concept of ferritin as a possible donor of iron to the mitochondria.

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria.

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions. The release process under optimized conditions (approx. 50 mumol/1 FMN, 1 mmol/l succinate, 0.35 mmol/1 Fe(III) (as ferritin iron), 37 degrees C and pH 7.40) amounts to 0.9--1.2 nmol iron/mg protein per min. The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.

Studies on the utilization of ferritin iron in the ferrochelatase reaction of isolated rat liver mitochondria.

The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found that isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 micromol/1 FMN, 37 degrees C, pH 7.4 and 0.5 mmol/l Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, amounted to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochrondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.

Reduction of exogenous FMN by isolated rat liver mitochondria. Significance to the mobilization of iron from ferritin.

When FMN is added to rat liver mitochondria or mitoplasts it is reduced at a rate of approx. 0.2 nmol . min-1 . mg-1 protein. Sonicated mitochondria do not reduce exogenous FMN. The reduction depends on drainage of reducing equivalents from the respiratory chain at the level of ubiquinone. The net production of reduced FMN is detectable only at oxygen concentrations less than 4-5 muM. The mitochondrial ubiquinol-FMN oxidoreductase provides a mechanism for the coupling of FMN-reduction to the reductive mobilization of iron from ferritin. The results are discussed in relation to the role of ferritin as a donor of iron to the mitochondria.

Decay kinetics of O2.- studied by direct spectrophotometry. Interaction with catalytic and non-catalytic substances.

The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.

Mobilization of iron from ferritin by isolated mitochondria. Effects of species compatibility between ferritin and mitochondria and iron content of ferritin.

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.

Effects of ferrous sulphate and non-ionic iron-polymaltose complex on markers of oxidative tissue damage in patients with inflammatory bowel disease.

BACKGROUND: Iron deficiency is a common complication of inflammatory bowel disease. Oral iron therapy may reinforce intestinal tissue injury by catalyzing production of reactive oxygen species. AIM: To compare the effects of ferrous sulphate and non-ionic iron-polymaltose complex on markers of oxidative tissue damage and clinical disease activity in patients with inflammatory bowel disease. METHODS: Forty-one patients with inflammatory bowel disease and iron deficiency were randomized to treatment with ferrous sulphate 100 mg twice a day or iron-polymaltose complex 200 mg once a day for 14 days. RESULTS: Following ferrous sulphate, plasma malondialdehyde increased (P = 0.02), while urine 8-isoprostaglandin F(2alpha) and plasma antioxidants did not change significantly. Iron-polymaltose complex did not change plasma malondialdehyde, urine 8-isoprostaglandin F(2alpha) or plasma antioxidants. Comparing the two treatments, changes in plasma malondialdehyde tended to differ (P = 0.08), while urine 8-isoprostaglandin F(2alpha) and plasma antioxidants did not differ. Neither ferrous sulphate nor iron-polymaltose complex altered clinical disease activity indices. CONCLUSIONS: Ferrous sulphate increased plasma malondialdehyde, a marker of lipid peroxidation. Comparing treatment with ferrous sulphate and iron-polymaltose complex, changes in plasma malondialdehyde tended to differ. Clinical disease activity was unchanged after both treatments.

Stimulated decay of superoxide caused by ferritin-bound copper.

The redox interaction between O2.- and ferritin cannot solely be regarded as as a Fe(II) release reaction. We demonstrate that native copper bound to horse spleen ferritin and apoferritin, stimulated the decay of O2.- in a catalytic reaction. Copper was determined by atomic absorption spectrophotometry. Decay of O2.- was monitored spectrophotometrically as the decrease in (A250-A360) at pH 9.5. The catalytic effect was linearly related to the copper content of the protein. Ferritin copper was less efficient than equimolar CuCl2, and iron-poor ferritin was more efficient than iron-rich ferritin. The results support a direct antioxidant function of ferritin.

Decay of superoxide catalyzed by ferritin.

Ferritin iron can be reduced by O2.-, released, and form a Fe(II)-chelator complex. However, the thermodynamic influence of the chelator may disturb the reaction balance. We therefore excluded the chelator and measured instead the effect of ferritin on the decay of O.2-, monitored by direct spectrophotometry at pH 9.5. Ferritin, but not apoferritin, accelerated the decay of O.2-. Ferritin iron was apparently the responsible agent. The effect of ferritin was maintained after several bursts of O.2-, and the ratio degraded O.2-/released Fe(II) greatly exceeded one, consistent with a catalytic reaction.

Sixty-minute normothermic liver ischemia in rats. Evidence that allopurinol improves liver cell energy metabolism during reperfusion but that timing of drug administration is important.

Allopurinol (ALL) improves energy metabolism in organs subjected to ischemia-reperfusion injury. The importance of different administration schedules of ALL has been studied in a rat liver model exposed to 60 min of normothermic ischemia followed by reperfusion. ALL (100 mg/kg) that administered in two doses, one prior to ischemia and one prior to reperfusion, improved production of adenosine triphosphate in the liver as well as bile flow during reperfusion. ALL administered in a single dose, either prior to ischemia or prior to reperfusion, was less effective. The concentration of hypoxanthine during ischemia increased in the groups given ALL prior to induction of ischemia. Based on the present findings, we argue that the beneficial effect of ALL administration can be potentiated by different drug-administration schedules. Our data also suggest that the prime mechanism of action for ALL is not only related to inhibition of free-oxygen-radicals production but that preservation of hypoxanthine, which can be used for ATP resynthesis and the scavenging properties of ALL itself, may be equally important.

Determination of manganese superoxide dismutase activity by direct spectrophotometry.

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O2- in a spectrophotometer, is described. Decay of O2- generated by KO2 at pH 9.5, was monitored as the fall in absorbance (A250nm-A360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O2- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu,Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu,Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.

Anemia in pregnancy in rural Tanzania: associations with micronutrients status and infections.

OBJECTIVE: We studied the association between anemia in pregnancy and characteristics related to nutrition and infections. DESIGN: Cross-sectional study. SETTING: Four antenatal clinics in rural northern Tanzania. SUBJECTS/METHODS: A total of 2547 women were screened for hemoglobin (Hb) and malaria plasmodia in capillary blood and for infections in urine. According to their Hb, they were assigned to one of five groups and selected accordingly, Hb<70 g/l (n=10), Hb=70-89 g/l (n=61), Hb=90-109 g/l (n=86), Hb=110-149 g/l (n=105) and Hb> or =150 g/l (n=50). The 312 selected subjects had venous blood drawn, were interviewed, and their arm circumference was measured. The sera were analyzed for ferritin, iron, total iron binding capacity (TIBC), cobalamin, folate, vitamin A, C-reactive protein (CRP), and lactate dehydrogenase (LD). Transferrin saturation (TFsat) was calculated. Urine was examined by dipsticks for nitrite. MAIN OUTCOME MEASURES: Unadjusted and adjusted odds ratio (OR and AOR) of anemia with Hb<90 g/l. RESULTS: Anemia (Hb<90 g/l) was associated with iron deficiency (low s-ferritin; AOR 3.4). The association with vitamin deficiencies were significant in unadjusted analysis (low s-folate; OR 3.1, low s-vitamin A; OR 2.6). Anemia was also associated with markers of infections (elevated s-CRP; AOR 3.5, urine nitrite positive; AOR 2.4) and hemolysis (elevated s-LD; AOR 10.1). A malaria positive blood slide was associated with anemia in unadjusted analysis (OR 2.7). An arm circumference less than 25 cm was associated with anemia (AOR 4.0). The associations with less severe anemia (Hb 90-109 g/l) were similar, but weaker. CONCLUSIONS: Anemia in pregnancy was associated with markers of infections and nutritional deficiencies. This should be taken into account in the management of anemia at antenatal clinics. SPONSORSHIP: The study was supported by the Norwegian Research Council (NFR) and the Centre for International Health, University of Bergen.

Anaemia in pregnancy: possible causes and risk factors in Nepali women.

OBJECTIVE: The aim of this study was to investigate the importance of nutritional deficiencies and infections in the development of anaemia in pregnant Nepali women. DESIGN: Case-control study. SETTING: Patan Hospital, Kathmandu, Nepal. SUBJECTS: A sub-sample (n=479) of all pregnant women (n=2856) coming for their first antenatal visit in a 12 month period, 1994-1995. Women who had already received any micronutrient supplementation (n=82), and those whose serum samples showed macroscopic haemolysis (n=7) were excluded. The remaining women (n=390) were included in the statistical analysis. They were divided into three groups; a non-anaemic control group, haematocrit (Hct)>33% (n=82), and two case-groups: moderately anaemic, Hct 25-33% (n=254), and severely anaemic, Hct<25% (n=54). RESULTS: We found high prevalences of nutritional deficiencies and intestinal infections, both among cases and controls. The prevalence of low s-ferritin was high, especially among the severely anaemic women (55.6%). In a multiple logistic regression model, the presence of low s-vitamin A, elevated s-C-reactive protein or hookworm infection was associated with a significantly increased risk of severe anaemia. The adjusted odds ratios (95% CI) were 8.38 (1.99, 35.30), 4.91 (1.22, 19.67) and 5.43 (1.20, 24.61), respectively. CONCLUSIONS: In addition to the present routine iron and folate supplementation to pregnant Nepali women, vitamin A supplementation needs to be considered. Prevention and treatment of infections should, together with dietary advice, be emphasized more strongly in the antenatal care. SPONSORSHIP: The Norwegian Research Council and the Norwegian Universities Committee for Development, Research and Education. European Journal of Clinical Nutrition (2000) 54, 3-8