Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.     

Effects of iron therapy on blood lead concentrations in infants

To determine whether blood lead concentration is elevated in iron-deficient infants, blood lead and serum ferritin concentrations, serum iron/transferring iron-binding capacity (Fe/TIBC) and complete blood counts were measured in 30 iron deficient and 35 control infants, aged 6–24 months. All 30 iron-deficient infants received iron supplementation (ferric hydroxide-polymaltose complex, 6 mg/kg Fe³⁺/day) for 1–6 months. Blood lead concentrations were measured in 18 of the iron deficient infants after their ferritin levels returned to the normal range. The geometric mean blood lead concentration was higher in iron deficient than in control infants (1.846 vs. 1.416 μg/dL). After iron therapy, the blood lead levels of iron-deficient infants decreased significantly compared with pre-treatment levels (1.785 vs. 2.386 μg/dL), and the hemoglobin and ferritin concentrations increased significantly. These findings indicate that iron deficiency increases blood lead concentrations in infants with very low blood lead concentrations.     

The effect of short-term exposure to low-iron diets on the mucosal processing of ionic iron

Short-term alterations in the amount of iron in the diets of rats caused substantial differences in the distribution of a test dose of radioiron between mucosal transferrin and mucosal ferritin, and also caused a change in the relative amounts of these two proteins in mucosal tissue without resulting in any detectable change in liver iron stores. These differences correlated with changes in the retention of radioiron by the intestinal mucosa and the transport of radioiron to the blood stream. These studies emphasize the importance of local changes in the intestinal mucosa in the regulation of dietary iron absorption.     

Iron metabolism in iron-deficient male quail

1. Male quails submitted 20 and 120 days to a low iron diet (7 ppm) were compared to female laying quails, exposed for 30 days to the same low iron regime, in order to compare the response of the iron metabolic control under a single (erythropoiesis) or a doubled (erythropoiesis and egg formation) iron demand. 2. Iron deposit in storage organs, the classical hematology and the intestinal iron absorption were analyzed in these animals. 3. In males, after 120 days, the iron deposits were reduced 50 and 75%, but hematological values (hematocrit and hemoglobin concentration) were normal, although in laying quails, after 30 days, an anemic condition was evident in both blood parameters and iron deposits, provoking an iron deficient erythropoiesis. 4. The enhancement of the intestinal iron uptake, confirms the anemic character of these birds.     

The association between iron metabolism with the change of blood pressure and risk of hypertension: A large cross-sectional study

BackgroundThe relationship between iron metabolism and variations in blood pressure and hypertension risk is still not clear. This study aimed to determine whether iron metabolism is associated with changes in blood pressure and hypertension prevalence in the general United States population.MethodsThe National Health and Nutrition Examination Survey (NAHNES) database contains data on 116876 Americans from 1999 to 2020 years. Data from the NHANES database were used to examine the relationships between iron metabolism (serum iron [SI], serum ferritin [SF], and soluble transferrin receptor [sTfR]) and changes in blood pressure and hypertension prevalence. Generalized linear models and restricted cubic spline (RCS) plot curves were used to estimate the relationship between iron metabolism and hypertension. Further, generalized additive models with smooth functions were used to identify the relationship between iron metabolism and blood pressure. Finally, a stratified subgroup analysis was performed.ResultsA total of 6710 participants were included in our analysis. The RCS plot showed a linear relationship between SI, as well as sTfR, and hypertension prevalence. SF and hypertension prevalence were associated in a J-shape. In addition, the relationship between SI and systolic blood pressure (SBP) and diastolic blood pressure (DBP) decreased initially and then increased. A correlation between SF, SBP, and DBP first decreased, then increased, and finally decreased. A positive linear correlation existed between sTfR and SBP, but it increased and then decreased with DBP.ConclusionThe correlation between SF and hypertension prevalence displayed a J-curve. In contrast, the correlation between SI, as well as sTfR, and hypertension risk was negative and positive, respectively.     

Thermoregulatory and acute-phase responses to endotoxin of full- term-pregnant rabbits

Fever, a fall in the plasma level of iron (Fe), and rises in the levels of certain plasma glycoproteins (indexed by protein-bound-N-acetylneuraminic acid [NANA]) normally occur during infection; they are thought to be mutually enhancing in host defense. It has been reported that fever is suppressed at full term of pregnancy; however, it is not known whether the blood chemical changes are similarly affected. Also, the mechanism for the suppression of fever is controversial. Since uteroplacental blood flow is at its maximum near term, competition between the demands of the fetoplacental unit and of thermoregulatory effectors might result in underperfusion of thermogenic tissues and therefore provide a basis for the lack of fever. To examine these questions, the changes in colonic temperature (Tco) and regional blood flow induced by Salmonella enteritidis endotoxin (LPS, 2 micrograms/kg iv) were compared in conscious nonpregnant and 30-day-pregnant rabbits 35 min after injection, using 15-microgram radiolabeled microspheres. In different rabbits, the effects of LPS on Tco and plasma Fe and NANA levels were measured before mating and at term. LPS induced fevers similar in heights and courses in both nonpregnant and full-term pregnant rabbits It caused decreases in the blood flows to brain, tongue, mammary gland, small intestine, and ear and increases in the blood flows to masseter muscle, bone, liver (hepatic artery), and left ventricle; blood flows to the kidneys, spleen, right ventricle, ovaries, and myometrium did not change. There were no significant differences in these vascular responses between nonpregnant and 30-day-pregnant rabbits, except a 28% reduction in the blood flow to the placentas.     

Folate Status Throughout Pregnancy and in Postpartum Period

The serial trends of the whole blood folate level in two groups of patients have been followed throughout pregnancy and up to six weeks postpartum. In those receiving iron alone the whole blood folate remained normal until the test at six weeks after delivery, at which time over half were in the deficient range. There appears to be a delay before this test reflects the current folate status when this changes rapidly. In those receiving iron plus 330 μg. of folic acid a day the results at this time were close to those at the beginning of pregnancy. Subnormal whole blood folate, red cell folate, and serum folate values occurred close to term in patients receiving iron alone, but were not found in those also receiving folic acid. Megaloblastic changes occurred at term in three patients receiving iron alone in whom the whole blood folate had repeatedly been low in early pregnancy.The observations are consistent with the previous suggestion that 300 μg. of folic acid daily is a suitable supplement to prevent deficiency in late pregnancy and the puerperium.     

复合疲劳对大鼠不同组织矿物质代谢影响研究

目的:研究复合疲劳大鼠血液、肌肉、肝脏和脑中矿物元素代谢变化的影响。方法:将30只大鼠随机分为正常对照组、食物限制组和复合疲劳组。经过5天的实验干预后,提取动物的血液、腓肠肌、肝脏和脑,并利用原子吸收分光光度法测量各组织中的钾(K)、钙(Ca)、镁(Mg)、铁(Fe)、锌(Zn)和铜(Cu)。结果:相对正常对照组和食物限制组,复合疲劳大鼠的肌肉、肝脏和脑中的K(P<0.01)和肝脏中的Fe(P<005)明显升高,血液中的Cu(与正常对照组比较P<0.01,与食物限制组比较P<0.05)明显下降;与对照组相比,复合疲劳大鼠的血液中的K明显升高(P<0.05),血液中的Mg和Zn(P<0.05),肌肉中的Ca、Mg和Zn(PCa<0.05,PMg<0.05,PZn<0.01),肝脏中的Ca和Zn(PCa<0.01,PZn<0.05),以及脑中的Fe、Mg和Zn(PFe<0.05,PMg<0.05,PZn<0.01)明显降低。结论:在复合疲劳状态下,大鼠血液、肌肉、肝脏和脑中的K、Ca、Mg、Fe、Zn和Cu代谢发生变化,可能在疲劳的发生与缓解中发挥作用。     

The valency state of absorbed iron appearing in the portal blood and ceruloplasmin substitution

Summary (1) Attempts to determine the redox-state of the absorbed iron, which appeared in the portal blood when the free iron-binding capacity was previously saturated, indicate that about 30–90% of this iron was in the ferrous state. This effect was particularly prominent after luminal administration of ferrous iron, but was also seen when iron was given in the ferric state. (2) Total iron absorption is significantly higher in ceruloplasmin-substituted copper-deficient animals as compared to copper-deficient controls. (3) The appearance rate of absorbed iron in the portal blood of copper-deficient animals increased several times immediately after the intravenous infusion of ceruloplasmin. (5) The distribution of absorbed iron was changed due to the ceruloplasmin substitution: it was increased in the reticulocytes (+66%), plasma (+400%) and the body (+ 112%), whereas in the liver it was decreased by about 78%. (5) In iron-deficient rats intravenously injected ceruloplasmin did not increase iron absorption. (6) The conclusion was drawn that, as for the entrance into the mucosa from the luminal side, also for the release at the contraluminal side into the portal blood, the ferrous state of iron is favoured and that ceruloplasmin accelerates the release into the portal blood by catalyzing the oxidation of ferrous iron due to its high Fe(II):oxygen oxidoreductase (EC 1.16.3.1) activity.     

Delta-aminolevulinate dehydratase activity in red blood cells of rats infected with Trypanosoma evansi

The aim of this study was to evaluate the activity of delta-aminolevulinate dehydratase (δ-ALA-D) in red blood cells of rats infected with Trypanosoma evansi and establish its association with haematocrit, serum levels of iron and zinc and lipid peroxidation. Thirty-six male rats (Wistar) were divided into 2 groups with 18 animals each. Group A was non-infected while Group B was intraperitoneally infected, receiving 7·5×106 trypomastigotes per animal. Each group was divided into 3 subgroups of 6 rats and blood was collected during different periods post-infection (p.i.) as follows: day 5 (A1 and B1), day 15 (A2 and B2) and day 30 PI (A3 and B3). Blood samples were collected by cardiac puncture to estimate red blood cell parameters (RBC), δ-ALA-D activity and serum levels of iron, zinc and thiobarbituric acid reactive substances (TBARS). Rats in group B showed a significant (P<0·05) reduction of RBC count, haemoglobin concentration and haematocrit at days 5 and 15 p.i. The activity of δ-ALA-D in blood was significantly (P<0·001) increased at days 15 and 30 p.i. δ-ALA-D activity in blood had a significant (P<0·05) negative correlation with haematocrit (r=-0·61) and haemoglobin (r=-0·70) at day 15 p.i. There was a significant (P<0·05) decrease in serum iron and zinc levels and an increase in TBARS levels (P<0·05) during infection. The δ-ALA-D activity in blood was negatively correlated with the levels of iron (r=-0·68) and zinc (r=-0·57) on day 30 p.i. It was concluded that the increased activity of δ-ALA-D in blood might have occurred in response to the anaemia in remission as heme synthesis was enhanced.     

Temporal changes in free iron levels after brain ischemia Relevance to the timing of iron chelation therapy in stroke

Whereas iron chelators have been proposed as therapeutic agents in stroke, changes in free iron levels have never been explored after focal brain ischemia. Therefore, free and total iron levels in cortical tissue and free iron levels in plasma were measured before and after (1, 4 and 24h) photothrombotic occlusion of cortical vessels in rats. Brain ferritin expression and localization were also investigated before and after (24, 72 and 192 h) occlusion. The results showed that free iron remained below detectable levels in plasma and that the lesion exhibited high levels of free and total iron. As compared to contralateral values, free iron levels in ischemic core and penumbra increased (+50%) at 1h and returned to control values at 4h post-occlusion. In contrast, the increase in total iron levels (+20-30%) was long-lasting, but confined to the ischemic core. A time-dependent increase in the expression of both chains of ferritin was detected in regions that previously exhibited free iron accumulation. Finally, ischemic damage was reduced by the liposoluble iron chelator 2,2'-dipyridyl (20 mg/kg, i.p.) when injected 15 min or 1 h post-occlusion, yet not later (4 h). In conclusion, our results show that focal brain ischemia results in an early and transient elevation in free iron levels in the ischemic tissue and suggest that free iron excess does not originate in blood. They also highlight the importance of starting iron chelation therapy as soon as possible after stroke.     

Effect of dietary iron deficiency on mineral levels in tissues of rats

To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats.     

The effect of Helicobacter pylori infection and dietary iron deficiency on host iron homeostasis: a study in mice

BACKGROUND: Helicobacter pylori, which requires iron to survive, may cause host iron deficiency by directly competing with the host for available iron or by impairing iron uptake as a consequence of atrophy-associated gastric hypochlorhydria. The aim of this study was to examine the effect of H. pylori infection and dietary iron deficiency on host iron homeostasis in a mouse model. MATERIALS AND METHODS: H. pylori SS1-infected and uninfected C57BL/6 mice, fed either a normal diet or an iron-deficient diet, were assessed for iron status and infection-associated gastritis over a 30-week period. RESULTS: After 10 weeks, serum ferritin values were higher in H. pylori-infected mice than in uninfected controls, irrespective of dietary iron intake (p = .04). The infection-related increase in body iron stores persisted in the iron-replete mice but diminished over time in mice with restricted dietary iron intake (p < .0001). At 30 weeks serum ferritin levels were lower in these animals (p = .063). No significant difference in bacterial numbers was detected at the 30-week time point (p > .05) and the histological changes observed were consistently associated with infection (p < .01) and not with the iron status of the mice (p = .771). CONCLUSIONS: Infection with H. pylori did not cause iron deficiency in iron-replete mice. However, diminished iron stores in mice as a result of limited dietary iron intake were further lowered by concurrent infection, thus indicating that H. pylori competes successfully with the host for available iron.     

Risk of iron overload is decreased in beating heart coronary artery surgery compared to conventional bypass.

Conventional cardiopulmonary bypass surgery (CCPB) increases the iron loading of plasma transferrin often to a state of plasma iron overload, with the presence of low molecular mass iron. Such iron is a potential risk factor for oxidative stress and microbial virulence. Here we assess 'off-pump' coronary artery surgery on the beating heart for changes in plasma iron chemistry. Seventeen patients undergoing cardiac surgery using the 'Octopus' myocardial wall stabilisation device were monitored at five time points for changes in plasma iron chemistry. This group was further divided into those (n=9) who had one- or two- (n=8) vessel grafts, and compared with eight patients undergoing conventional coronary artery surgery. Patients undergoing beating heart surgery had significantly lower levels of total plasma non-haem iron, and a decreased percentage saturation of their transferrin at all time points compared to conventional bypass patients. Plasma iron overload occurred in only one patient undergoing CCPB. Beating heart surgery appears to decrease red blood cell haemolysis, and tissue damage during the operative procedures and thereby significantly decreases the risk of plasma iron overload associated with conventional bypass.     

Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women.

Iron deficiency anemia (IDA) during pregnancy continues to be of world-wide concern. IDA is a risk factor for preterm delivery and subsequent low birth weight, and possibly for poor neonatal health. Iron supplementation in pregnancy is a widely recommended practice, yet intervention programs have met with many controversies. In our study, 300 women at different trimesters of pregnancy were enrolled in a trial of oral administration of ferrous sulfate (520 mg once a day) or 30% iron-saturated bovine lactoferrin (bLf) (100 mg twice a day). Pregnant women refusing treatment represented the control group. In this group hemoglobin and total serum iron values measured after 30 d without treatment decreased significantly, especially in women at 18-31 weeks of pregnancy. In contrast, after 30 d of oral administration of bLf, hemoglobin and total serum iron values increased and to a greater extent than those observed in women treated orally for 30 d with ferrous sulfate, independently of the trimester of pregnancy. Unlike ferrous sulfate, bLf did not result in any side effects. These findings lead us to hypothesize that lactoferrin could influence iron homeostasis directly or through other proteins involved in iron transport out of the intestinal cells into the blood.     

内耳形态学和生理指标对缺铁性肾虚耳聋大鼠模型的评价作用

目的探讨耳蜗显微结构和生理指标对缺铁性肾虚耳聋大鼠模型的评价作用。方法选用体重30～32 g、无耳疾、听性脑干反应(auditory brainstem response,ABR)阈值正常的1～2周龄SPF级SD大鼠120只,雌雄分养,分为缺铁组80只、正常对照组40只,饲养时间12周;以出现肾虚证和至少一耳ABR阈值≥15 dB,作为判断肾虚耳聋的标准,获得缺铁性肾虚耳聋大鼠22只,从中选取肾虚耳聋大鼠20只,同时以20只正常大鼠作对照。观察耳蜗血管纹、螺旋器等耳蜗显微结构变化,检测ABR阈值以及血红蛋白和血清铁等指标的变化。结果实验组和正常对照大鼠的血红蛋白和血清铁分别为11.80 g/L,4.5μmol/L和45.9 g/L,22.23μmol/L,ABR阈值分别为(30±5)dB和(10±5)dB;实验组血管纹血管明显减少;螺旋器毛细胞听毛有缺失、倒伏现象。结论缺铁性肾虚耳聋大鼠血红蛋白、血清铁和ABR阈值,以及耳蜗血管纹、螺旋器等耳蜗显微结构变化等指标,均较为稳定,是较好的评价指标。     

Changes in the level of sulfhydryl groups in the organs and blood of mice administered a highly dispersed iron powder against a background of developing anemia

Alimentary iron deficiency causes significant changes in thiol content: the increase in the content of protein sulfhydryl groups in organs at late stages and the decrease in that of non-protein sulfhydryl groups at early stages of experiment. A preliminary introduction of 10 mg/kg fine iron powder (FIP) into animals fed iron-free food leads to the decrease in sulfhydryl group content in organs and blood of experimental mice. The decrease in sulfhydryl group content at FIP introduction might be connected with changes in activity of glutathione-dependent enzymes.     

Protection by Desferrioxamine Against Histopathological Changes of the Liver in the Post-Oligaemic Phase of Clinical Haemorrhagic Shock in Dogs: Correlation with Improved Survival Rate and Recovery

Haemorrhagic shock was produced in anaesthetized dogs, by rapid arterial bleeding to mean arterial blood pressure 35 mmHg, and maintained oligaemic for 4 h followed by return of withdrawn blood(R0WB). Dogs were observed for 72 h after ROWB for survival and recovery, and, for histopathological (HP) studies on liver, dogs were sacrificed 2 h after ROWB in non-survival experiments. Desferrioxamine mesylate (25mg/kg) was administered intra-muscularly at 2,3 and 4h after blood loss in survival experiments and for HP studies the drug was given at 4 h in one group and at 2 h plus 4 h after blood loss in the second group. With the drug given at 3 or 4h, survival was 70% and 100% while in the 2h and the untreated groups it was 50%. Recovery was rapid in all the drug treated survivors, few became conscious within 30min. showed slight activity by 4-6 h, all were almost normally active by 24 and fully so by 72 h after ROWB. All the 5 control survivors remained unconscious/drowsy upto 24 h; 3 were sluggish at 72 h. By group analysis, serum iron elevation during the oligaemic and at the end of the post-oligaemic phase was less in the drug-treated animals. HP changes of shock in the liver studied by light microscopy, were markedly reduced in severity and were less prevalent in the drug-treated dogs. The salutory effects of desferrioxamine may be due to inhibition of iron catalyzed free-radical production and tissue damage, through its strong iron chelating action. It may have a therapeutic advantage in this emergency condition without the disadvantages of toxicity inherent in prolonged use.     

Mobilization of storage iron is reflected in the iron isotopic composition of blood in humans

We recently showed in an animal model that iron isotopic composition varies substantially between different organs. For instance, iron in ferritin-rich organs—such as the major storage tissues liver, spleen, and bone marrow—contain a larger fraction of the heavy iron isotopes compared with other tissues, including blood. As a consequence, partitioning of body iron into red blood cells and storage compartments should be reflected in the isotopic pattern of blood iron. To confirm this hypothesis, we monitored blood iron isotope patterns in iron-overloaded subjects undergoing phlebotomy treatment by multicollector inductively coupled plasma mass spectrometry. We found that bloodletting and consequential replacement of lost blood iron by storage iron led to a substantial increase of the heavy isotope fraction in the blood. The progress of iron depletion therapy and blood loss was quantitatively traceable by isotopic shifts of as much as +1‰ in δ(⁵⁶Fe). These results show that—together with iron absorption efficiency—partitioning of iron between blood and iron storage tissues is an important determinant of blood iron isotopic patterns, which could make blood iron isotopic composition the first composite measure of iron metabolism in humans.