Changes in cerebellar iron metabolism and their prevention by allopurinol in oxidative stress related to acute ethanol administration in rats

An acute ethanol load results in an increased total nonheme iron and cytosolic low molecular weight iron content in rat cerebellum. At the same time the cerebellar susceptibility to lipid peroxidation is enhanced whereas the alpha-tocopherol and ascorbate content is decreased. These changes argue for a cerebellar ethanol-induced oxidative stress, which is prevented by prior allopurinol administration.     

Visualization of non-heme ferric and ferrous iron by highly sensitive non-heme iron histochemistry in the stress-induced acute gastric lesions in the rat

Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.     

Zinc, Copper, Iron, and Chromium Concentrations in Young Patients with Type 2 Diabetes Mellitus

Homeostasis of trace elements can be disrupted by diabetes mellitus. On the other hand, disturbance in trace element status in diabetes mellitus may contribute to the insulin resistance and development of diabetic complications. The aim of present study was to compare the concentration of essential trace elements, zinc, copper, iron, and chromium in serum of patients who have type 2 diabetes mellitus (n = 20) with those of nondiabetic control subjects (n = 20). The serum concentrations of zinc, copper, iron, and chromium were measured by means of an atomic absorption spectrophotometer (Shimadzu AA 670, Kyoto, Japan) after acid digestion. The results of this study showed that the mean values of zinc, copper, and chromium were significantly lower in the serum of patients with diabetes as compared to the control subjects (P < 0.05). Our results show that deficiency of some essential trace elements may play a role in the development of diabetes mellitus.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Viral RNA kinetics is associated with changes in trace elements in target organs of Coxsackie virus B3 infection

Trace elements are pivotal for the host defense, as well as potentially important for viral replication and virulence. Studies of sequential changes in viral replication in target organs of infection are sparse and a possible association with changes in specific trace elements is unknown. In this study Balb/c mice were infected with Coxsackie virus B3 (CVB3). Results indicated that sequential changes in viral replication (RT-PCR) were related to changes in trace element (arsenic, copper, iron, selenium and zinc) concentrations (as determined by ICP-MS) on days 3, 5 and 7 of the infection in serum, heart, lung, liver, pancreas, kidney, spleen, intestine and brain. After an initial viral peak on day 3, viral load drastically decreased in all organs, i.e. by >99% (serum), 97% (lung), 98% (liver), 60% (pancreas), 95% (kidney) and 93% (spleen), except in the heart, intestine and brain in which viral load increased after day 3. Selenium decreased in all organs except the heart while arsenic decreased in all organs except the kidney, spleen and brain. Moreover, selenium was negatively correlated to viral load in serum, liver, pancreas and intestine. To conclude, these findings give evidence that trace elements are directly involved in the replication of CVB3.     

Effects of EDTA on trace elements and cardiovascular function in the anesthetised rabbit

The present study was undertaken in order to study the effects of the broad-acting chelating agent CaNa₂-EDTA on plasma trace elements and cardiovascular function in anesthetised New Zealand White rabbits. Trace elements are important for cardiovascular and immune functions and the rabbit is a well-accepted species in cardiovascular studies. The test compound CaNa₂-EDTA was administered intravenously to rabbits at single doses of 4, 20, and 100 mg/kg. In addition, at 20 mg/kg, the effects of a second dose after 3 h were also investigated. Heart rate, blood pressure and body temperature were continuously monitored during a 6-h interval after injection of CaNa₂-EDTA. Immediately before administration (−1 min) and at 3 and 6 h over the period of the experiment, the plasma cytokine response (tumor necrosis factor-α) and trace elements (Mn, Cu, Zn, Se, Ag, Cd, Hg, Pb) were measured. Regardless of dose, blood pressure was found to decrease, but no corresponding changes in heart rate were observed. Both a repeated administration of 20 mg/kg and a single dose of 100 mg/kg were detrimental and caused severe cardiovascular effects and lethality. α-TNF tended to increase, though only at 100 mg/kg. The electrocardiogram and body temperature were not affected by the treatment. The most pronounced trace element change was a dose-dependent increase in Mn that was equally pronounced at all time-points after the dose. There was an initial decrease in Cd at low dose levels (4 and 20 mg/kg) that turned into an increase after 6 h at 20 mg/kg and from 2 h at 100 mg/kg. A similar pattern with pronounced decreases at low dose levels was observed for Zn. Cu decreased similarly at all dose levels. For the other trace elements, no or inconsistent effects were observed. This model allows the study of concomitant cardiovascular and trace element changes during treatment with drugs and chelating agents preceding a possible lethal end point and associated pathophysiologic changes.     

Changes in free radicals,trace elements,and neurophysiological function in rats with liver damage induced byD-galactosamine

The changes in trace elements, free radicals, and neurophysiological function were investigated in rats with liver damage induced byd-galactosamine (GalN). The elevated results showed that all the parameters related to free radical metabolism changed after administration of GalN. Relative free radical concentration, malonaldehyde (MDA), and oxidized glutathione (GSSG) elevated, but reduced glutathione (GSH) decreased. Concurrently, zinc, copper, manganese, and selenium contents in liver were significantly reduced, whereas iron was elevated. In rats with hepatic encephalopathy (HE) owing to fulminant hepatic failure (FHF) induced by a high dosage of GalN, the latencies of VEPs were delayed. Moreover, there is a correlation between Zn content of brain and the latencies of VEPs. The results of this study suggested that lipid peroxidation by free radicals might be responsible for GalN-induced liver damage in which trace elements were involved, and that change in brain Zn might play a role in the neural inhibition of HE owing to FHF.     

Gastrointestinal uptake of trace elements are changed during the course of a common human viral (Coxsackievirus B3) infection in mice.

Most infectious diseases are accompanied by a change in levels of several trace elements in the blood. However, it is not known whether changes in the gastrointestinal uptake of trace elements contribute to this event. Coxsackievirus B3 (CVB3), adapted to Balb/c mice, was used to study whether infection induces gene expression of metallothionein (MT1) and divalent-metal transporter 1 (DMT1) in the intestine and liver and hepcidin in the liver, as well as whether trace elements in these tissues are changed accordingly. Quantitative expression of CVB3, MT1, DMT1 and hepcidin was measured by real-time RT-PCR and six trace elements by ICP-MS on days 3, 6 and 9 of the infection. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. High concentrations of virus were found in the intestine and liver on day 3 and in the intestine on day 6. MT1 in the intestine and liver increased on days 3 and 6. The increase of MT1 in the liver correlated positively with Cu and Zn. Hepcidin in the liver showed a non-significant increase on days 3 and 6 of the infection, whereas DMT1 in the intestine decreased on day 9. Accordingly, iron (Fe) in the liver increased progressively during the disease, whereas in the intestine DMT1 was negatively correlated to Fe. Arsenic (As), cadmium (Cd) and mercury (Hg) were found to decrease to various degrees in the intestine, serum and liver. Thus, enteroviral infections, and possibly many other infections, may cause a change in the gastrointestinal uptake of both non-essential and essential trace elements.     

Iron accumulation with age, oxidative stress and functional decline

Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength) in male Fischer 344 X Brown Norway rats fed ad libitum (AL) or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age) at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR) rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.     

The nitric oxide complex of ferrous soybean lipoxygenase-1. Substrate, pH, and ethanol effects on the active-site iron

Soybean lipoxygenase is a non-heme iron enzyme that catalyzes the hydroperoxidation of linoleic acid by dioxygen. Exposure of ferrous lipoxygenase to nitric oxide yields a species displaying an electron paramagnetic resonance spectrum characteristic of a nearly axial S = 3/2 electronic spin system arising from the ferrous-nitrosyl complex. That spectrum is pH-sensitive, reflecting changes in the environment of the metal ion between pH 7 and 11. Addition of ethanol abolishes the effects of pH in a saturable fashion, resulting in a spectrum similar to that seen at pH 7. Exchange of lipoxygenase into H2(17)O leads to no significant line broadening in the low field portion of the spectrum, suggesting no coordination of water. The ferrous enzyme displays greater affinity for NO at pH 9 (where the enzyme is most active) than at pH 7. The binding of linoleic acid is competitive with that of NO at pH 9, but not at pH 7. These results are interpreted in terms of a model including only one iron site for exogenous ligands and an otherwise relatively stable iron coordination environment.     

Effect of antiperoxidative drugs on gastric damage induced by ethanol in rats

Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl radicals, contributes to lipid peroxidation and lesion formation in the gastric mucosa after absolute ethanol administration.     

Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective

Individuals who carry the most active alcohol dehydrogenase (ADH) isoforms are protected against alcoholism. This work addresses the mechanism by which a high ADH activity leads to low ethanol intake in animals. Male and female ethanol drinker rats (UChB) were allowed access to 10% ethanol for 1 h. Females showed 70% higher hepatic ADH activity and displayed 60% lower voluntary ethanol intake than males. Following ethanol administration (1 g/kg ip), females generated a transient blood acetaldehyde increase ("burst") with levels that were 2.5-fold greater than in males (P < 0.02). Castration of males led to 1) an increased ADH activity (+50%, P < 0.001), 2) the appearance of an acetaldehyde burst (3- to 4-fold vs. sham), and 3) a reduction of voluntary ethanol intake comparable with that of na?ve females. The ADH inhibitor 4-methylpyrazole blocked the appearance of arterial acetaldehyde and increased ethanol intake. Since the release of NADH from the ADH.NADH complex constitutes the rate-limiting step of ADH (but not of ALDH2) activity, endogenous NADH oxidizing substrates present at the time of ethanol intake may contribute to the acetaldehyde burst. Sodium pyruvate given at the time of ethanol administration led to an abrupt acetaldehyde burst and a greatly reduced voluntary ethanol intake. Overall, a transient surge of arterial acetaldehyde occurs upon ethanol administration due to 1) high ADH levels and 2) available metabolites that can oxidize hepatic NADH. The acetaldehyde burst is strongly associated with a marked reduction in ethanol intake.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

The relationship between human cytomegalovirus infection and atherosclerosis development

Transport characteristics of essential trace elements as zinc, copper, selenium and iron have been studied in maternal–fetal direction in normal pregnancies, using in vitro perfusion of human placental lobules. Solutions of trace elements corresponding to twice the physiological concentrations were injected (100 l bolus) into the maternal arterial perfusate. Serial perfusate samples were collected every 30 sec from venous outflows for a study period of 5 min. Concentrations of these trace elements and their transport kinetics were determined. Transport fractions (TF) of zinc, copper, selenium and iron averaged 0.21, 0.49, 0.55 and 0.10% of maternal load respectively. Other parameters such as area under the curve, clearance, elimination constant, absorption and elimination rates showed some significant differences between the various elements. Copper and selenium appear to be transported passively in maternal–fetal direction, while for iron and zinc, role of active transport for transfer across the human placental membrane cannot be discounted. We speculate that alterations in copper: zinc TR50 (transport rate for 50% efflux) and TF ratios could serve as useful indicators for assessing placental transport status of these essential elements in complicated pregnancy states.     

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Evaluation of the Relationship Between Height and Zinc,Copper, Iron,Calcium, and Magnesium Levels in Healthy Young Children in Beijing,China

Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals’ concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al’s reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.     

A combination of ischemic preconditioning and allopurinol protects against ischemic injury through a nitric oxide-dependent mechanism

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50 mg/kg) was intraperitoneally administered 18 and 1 h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5 h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

Non-heme Iron as Ferrous Sulfate Does Not Interact with Heme Iron Absorption in Humans

The absorption of heme iron has been described as distinctly different from that of non-heme iron. Moreover, whether heme and non-heme iron compete for absorption has not been well established. Our objective was to investigate the potential competition between heme and non-heme iron as ferrous sulfate for absorption, when both iron forms are ingested on an empty stomach. Twenty-six healthy nonpregnant women were selected to participate in two iron absorption studies using iron radioactive tracers. We obtained the dose?Cresponse curve for absorption of 0.5, 10, 20, and 50?mg heme iron doses, as concentrated red blood cells. Then, we evaluated the absorption of the same doses, but additionally we added non-heme iron, as ferrous sulfate, at constant heme/non-heme iron molar ratio (1:1). Finally, we compare the two curves by a two-way ANOVA. Iron sources were administered on an empty stomach. One factor analysis showed that heme iron absorption was diminished just by increasing total heme iron (P?<?0.0001). The addition of non-heme iron as ferrous sulfate did not have any effect on heme iron absorption (P?=?NS). We reported evidence that heme and non-heme iron as ferrous sulfate does not compete for absorption. The mechanism behind the absorption of these iron sources is not clear.