Urinary iron excretion for evaluating iron chelation efficacy in children with thalassemia major.

BACKGROUND: In patients with thalassemia major, examination routinely used for the evaluation of iron load in Indonesia is serum ferritin, but it is strongly influenced by other factors such as infections, inflammation and vitamin C levels. Evaluation of urinary iron excretion is an important and easy method to indicate iron chelation efficacy. OBJECTIVE: To determine the efficacy of iron chelation therapy by urinary iron examination and to evaluate its correlation with the time of transfusion, serum ferritin level, transferrin saturation and T2* MRI. METHODS: Prospective cohort study was conducted in children with thalassemia major aged 7-<18 years old who received DFP therapy. Twenty-four-hour urine collections were examined through inductively coupled plasma - mass spectrometry (ICP-MS). Patient's serum ferritin, transferrin saturation, peripheral blood, differential count and T2* MRI was documented during the study. Data analysis is based on urine iron level, body iron balance and the correlation between urine iron level, serum ferritin, transferrin saturation and T2* MRI and dosage of DFP. RESULTS: Thirty (55%) subjects showed a higher urine iron level on the day prior to transfusion (mean: 12,828 SD ±12,801 µg/24 h) in comparison to post transfusion (mean: 10,985 SD ±10,023 µg/24 h). All subjects had positive iron balance (mean 524 SD ±230 mg). There were positive correlation between urine iron level and transferrin saturation (r = 0.559, p = 0.01) and serum ferritin (r = 0.291, p = 0.03), no correlation found with T2* MRI results. CONCLUSIONS: There is a relationship to urinary iron excretion in response to chelation therapy and the degree of iron load.

Therapeutic mechanism of combined oral chelation therapy to maximize efficacy of iron removal in transfusion-dependent thalassemia major - a pilot study.

OBJECTIVES: Three iron chelators are used to treat transfusion-dependent beta-thalassemia: desferrioxamine (DFO), deferasirox (DFX), and deferiprone (DFP). Compliance is low for DFO as it cannot be administered orally. Combined administration of DFP and DFX is orally available, however, the therapeutic mechanism is unknown. This pilot study investigated the iron removal mechanisms of DFX and DFP treatment in patients with transfusion-dependent thalassemia major. METHODS: Each patient received three treatments sequentially: (1) DFX monotherapy, (2) DFP monotherapy, and (3) DFX/DFP combination therapy with a four-day washout period between each treatment. Urine and stool specimens were collected to determine the primary outcome of iron excretion volumes. RESULTS: The mean iron excretion was seven times higher after combination therapy with DFX and DFP. Monotherapies also increased excretions volumes, though to a significantly lesser degree. Combined administration of DFX and DFP achieves maximum iron removal in transfusion-dependent thalassemia major compared to monotherapy with either drug. CONCLUSIONS: We suggest combination therapy in chronic severe iron overload cases, especially for patients in poor compliance with DFO/DFP combination therapy or those exhibiting poor iron removal from DFX or DFP monotherapy.

Iron handling by the human kidney: glomerular filtration and tubular reabsorption both contribute to urinary iron excretion.

In physiological conditions, circulating iron can be filtered by the glomerulus and is almost completely reabsorbed by the tubular epithelium to prevent urinary iron wasting. Increased urinary iron concentrations have been associated with renal injury. However, it is not clear whether increased urinary iron concentrations in patients are the result of increased glomerular iron filtration and/or insufficient tubular iron reabsorption and if these processes contribute to renal injury. We measured plasma and urine iron parameters and urinary tubular injury markers in healthy human subjects ( n = 20), patients with systemic iron overload ( n = 20), and patients with renal tubular dysfunction ( n = 18). Urinary iron excretion parameters were increased in both patients with systemic iron overload and tubular dysfunction, whereas plasma iron parameters were only increased in patients with systemic iron overload. In patients with systemic iron overload, increased urinary iron levels were associated with elevated circulating iron, as indicated by transferrin saturation (TSAT), and increased body iron, as suggested by plasma ferritin concentrations. In patients with tubular dysfunction, enhanced urinary iron and transferrin excretion were associated with distal tubular injury as indicated by increased urinary glutathione S-transferase pi 1-1 (GSTP1-1) excretion. In systemic iron overload, elevated urinary iron and transferrin levels were associated with increased injury to proximal tubules, indicated by increased urinary kidney injury marker 1 (KIM-1) excretion. Our explorative study demonstrates that both glomerular filtration of elevated plasma iron levels and insufficient tubular iron reabsorption could increase urinary iron excretion and cause renal injury.

RNA oxidation and iron levels in patients with diabetes.

AIM: The urinary biomarker for oxidative stress to RNA, 8-oxo-7,8-dihydro-guanosine (8-oxoGuo) is associated with mortality in patients with type 2 diabetes. Iron has also been linked to diabetes. In individuals with untreated hereditary iron overload it has been observed that 8-oxoGuo was higher compared to controls. In the current study, we hypothesized that 8-oxoGuo was associated with diagnosis of diabetes, and that iron confounded this association. METHODS: Participants from a general Danish population were included in the study (n = 3567). UPLC-MS/MS method was used for 8-oxoGuo (nmol/mmol creatinine) measurement in spot urine. Iron biomarkers included total plasma iron, ferritin, transferrin saturation (TS) and transferrin. RESULTS: 8-oxoGuo was 17% higher in diabetes patients (n = 208) compared to non-diabetes controls. Unadjusted logistic regression model showed an odds ratio of diabetes of 1.38 (95%CI:1.21-1.57, P < 0.0001) per unit increase of 8-oxoGuo. When the model was adjusted for possible confounders the odds ratio was 1.09 (95%CI:0.94-1.26, P = 0.24). When additional adjustment was performed including ferritin, TS, or transferrin, respectively, the OR were 1.14 (95%CI:0.97-1.33, P = 0.09), 1.10 (95%CI: 0.95-1.28, P = 0.18), and 1.17 (95%CI:1.01-1.38, P = 0.04). CONCLUSIONS: Our study indicates that 8-oxoGuo is higher in diabetes patients. The lack of association between 8-oxoGuo and diabetes in the adjusted model may be due to the cross-sectional design including post-treatment bias. Our data did not show consistent effect of all iron biomarkers in relation to diabetes. Most likely, the iron biomarkers were affected by inflammation thus not reflecting true iron levels.

Modulation of urinary siderophores by the diet, gut microbiota and inflammation in mice.

Mammalian siderophores are believed to play a critical role in maintaining iron homeostasis. However, the properties and functions of mammalian siderophores have not been fully clarified. In this study, we have employed Chrome Azurol S (CAS) assay which is a well-established method for bacterial siderophores study, to detect and quantify mammalian siderophores in urine samples. Our study demonstrates that siderophores in urine can be altered by diet, gut microbiota and inflammation. C57BL/6 mice, fed on plant-based chow diets which contain numerous phytochemicals, have more siderophores in the urine compared to those fed on purified diets. Urinary siderophores were up-regulated in iron overload conditions, but not altered by other tested nutrients status. Further, germ-free mice displayed 50% reduced urinary siderophores, in comparison to conventional mice, indicating microbiota biotransformation is critical in generating or stimulating host metabolism to create more siderophores. Altered urinary siderophores levels during inflammation suggest that host health conditions influence systemic siderophores level. This is the first report to measure urinary siderophores as a whole, describing how siderophores levels are modulated under different physiological conditions. We believe that our study opens up a new field in mammalian siderophores research and the technique we used in a novel manner has the potential to be applied to clinical purpose.

Iron excretion in iron dextran-overloaded mice.

BACKGROUND: Iron homeostasis in humans is tightly regulated by mechanisms aimed to conserve iron for reutilisation, with a negligible role played by excretory mechanisms. In a previous study we found that mice have an astonishing ability to tolerate very high doses of parenterally administered iron dextran. Whether this ability is linked to the existence of an excretory pathway remains to be ascertained. MATERIALS AND METHODS: Iron overload was generated by intraperitoneal injections of iron dextran (1 g/kg) administered once a week for 8 weeks in two different mouse strains (C57bl/6 and B6D2F1). Urinary and faecal iron excretion was assessed by inductively coupling plasma-mass spectrometry, whereas cardiac and liver architecture was evaluated by echocardiography and histological methods. For both strains, 24-hour faeces and urine samples were collected and iron concentration was determined on days 0, 1 and 2 after iron administration. RESULTS: In iron-overloaded C57bl/6 mice, the faecal iron concentration increased by 218% and 157% on days 1 and 2, respectively (p<0.01). The iron excreted represented a loss of 14% of total iron administered. Similar but smaller changes was also found in B6D2F1 mice. Conversely, we found no significant changes in the concentration of iron in the urine in either of the strains of mice. In both strains, histological examination showed accumulation of iron in the liver and heart which tended to decrease over time. CONCLUSIONS: This study indicates that mice have a mechanism for removal of excess body iron and provides insights into the possible mechanisms of excretion.

A prospective study of tubular dysfunction in pediatric patients with Beta thalassemia major receiving deferasirox.

BACKGROUND: Beta thalassemia major is a lifelong transfusion-dependent disorder. Transfusion-dependent thalassemia patients are prone to develop renal dysfunction due to iron overload, chronic anemia, and/or chelation therapy. METHODS: In this prospective study, thalassemia patients who fitted inclusion and exclusion criteria received Deferasirox 20 mg/kg/day. A complete biochemistry analysis of serum and 24-hour-urine specimens was performed before and after treatment. Estimated glomerular filtration rate (eGFR), Fractional excretion of sodium (FENA), potassium (FEK), uric acid (FEUA), and the maximum ratio of tubular reabsorption of phosphorus to eGFR (TmP/GFR) at baseline and after treatment was calculated and compared. RESULTS: A total of 30 patients with mean age of 4.9 ± 3.2 years were recruited. The mean serum creatinine increased significantly after 6 months of treatment (0.54 ± 0.08 vs. 0.67 ± 0.16, P < .001) while eGFR was decreased (104.36 ± 19.62 vs. 86.00 ± 16.92, P < .001). Mean potassium level in serum was increased after treatment, while serum calcium, magnesium, and uric acid levels decreased significantly (P > .05). A significant increase was confirmed for mean urinary ß2-microglobulin (ß2-MG), protein, uric acid, calcium, and magnesium (P > .05). CONCLUSION: Our findings highlighted tubular nephropathy induced by Deferasirox in patients with beta thalassemia, and confirmed the necessity for diligent monitoring of renal function in thalassemia patients receiving Deferasirox.

Hepcidin expression in iron overload diseases is variably modulated by circulating factors.

Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.

Timed non-transferrin bound iron determinations probe the origin of chelatable iron pools during deferiprone regimens and predict chelation response.

BACKGROUND: Plasma non-transferrin bound iron refers to heterogeneous plasma iron species, not bound to transferrin, which appear in conditions of iron overload and ineffective erythropoiesis. The clinical utility of non-transferrin bound iron in predicting complications from iron overload, or response to chelation therapy remains unproven. We undertook carefully timed measurements of non-transferrin bound iron to explore the origin of chelatable iron and to predict clinical response to deferiprone. DESIGN AND METHODS: Non-transferrin bound iron levels were determined at baseline and after 1 week of chelation in 32 patients with thalassemia major receiving deferiprone alone, desferrioxamine alone, or a combination of the two chelators. Samples were taken at baseline, following a 2-week washout without chelation, and after 1 week of chelation, this last sample being taken 10 hours after the previous evening dose of deferiprone and, in those receiving desferrioxamine, 24 hours after cessation of the overnight subcutaneous infusion. Absolute or relative non-transferrin bound iron levels were related to transfusional iron loading rates, liver iron concentration, 24-hour urine iron and response to chelation therapy over the subsequent year. RESULTS: Changes in non-transferrin bound iron at week 1 were correlated positively with baseline liver iron, and inversely with transfusional iron loading rates, with deferiprone-containing regimens but not with desferrioxamine monotherapy. Changes in week 1 non-transferrin bound iron were also directly proportional to the plasma concentration of deferiprone-iron complexes and correlated significantly with urine iron excretion and with changes in liver iron concentration over the next 12 months. CONCLUSIONS: The widely used assay chosen for this study detects both endogenous non-transferrin bound iron and the iron complexes of deferiprone. The week 1 increments reflect chelatable iron derived both from liver stores and from red cell catabolism. These increments correlate with urinary iron excretion and the change in liver iron concentration over the subsequent year thus predicting response to deferiprone-containing chelation regimes. This clinical study was registered at clinical.trials.gov with the number NCT00350662.

Dysmetabolic hyperferritinemia is associated with normal transferrin saturation, mild hepatic iron overload, and elevated hepcidin.

Hyperferritinemia is common in individuals with the metabolic syndrome (dysmetabolic hyperferritinemia), but its pathophysiology and the degree to which it reflects tissue iron overload remains unclear. We conducted a cross-sectional study evaluating ten cases with dysmetabolic hyperferritinemia for liver iron overload and compared their serum iron indices and urine hepcidin levels to healthy controls. Seven out of ten cases had mild hepatic iron overload by magnetic resonance imaging (MRI) (median, 75 micromol/g dry weight). Cases had higher serum ferritin than controls (median, 672 microg/L vs. 105 microg/L, p < 0.001), but the median transferrin saturation was not significantly different (38% vs. 36%, p = 0.5). Urinary hepcidin was elevated in dysmetabolic hyperferritinemia (median; 1,584 g/mg of creatinine vs. 799 ng/mg of creatinine, p = 0.05). Dysmetabolic hyperferritinemia is characterized by hyperferritinemia with normal transferrin saturation, elevated hepcidin levels, and mild liver iron overload in a subset of patients.

Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload.

BACKGROUND/AIMS: Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS: Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS: No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS: Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).

Hepcidin in iron overload disorders.

Hepcidin is the principal regulator of iron absorption in humans. The peptide inhibits cellular iron efflux by binding to the iron export channel ferroportin and inducing its internalization and degradation. Either hepcidin deficiency or alterations in its target, ferroportin, would be expected to result in dysregulated iron absorption, tissue maldistribution of iron, and iron overload. Indeed, hepcidin deficiency has been reported in hereditary hemochromatosis and attributed to mutations in HFE, transferrin receptor 2, hemojuvelin, and the hepcidin gene itself. We measured urinary hepcidin in patients with other genetic causes of iron overload. Hepcidin was found to be suppressed in patients with thalassemia syndromes and congenital dyserythropoietic anemia type 1 and was undetectable in patients with juvenile hemochromatosis with HAMP mutations. Of interest, urine hepcidin levels were significantly elevated in 2 patients with hemochromatosis type 4. These findings extend the spectrum of iron disorders with hepcidin deficiency and underscore the critical importance of the hepcidin-ferroportin interaction in iron homeostasis.

Bone metabolism and oxidative stress in postmenopausal rats with iron overload.

Osteoporosis is associated with many etiological causes such as nutrition, cytokines, hormones, and aging. Recently, reactive oxygen species (ROS) are considered to be responsible for the aging process and osteoporosis. We investigated the relationship between ROS and bone metabolism in young female and postmenopausal rats, by using dietary iron overload and several indices including bone metabolic markers, oxidative stress and antioxidant markers, and cytokines. Postmenopausal rats exhibited significant decreases in serum alkaline phosphatase activity and the level of osteocalcin as bone formation markers compared with young female rats; however, urinary excretion of deoxypyridinoline, a bone resorption marker, did not change. On the other hand, a 5% iron lactate diet for 4 weeks in postmenopausal rats led to significantly increased excretion of urinary deoxypyridinoline and 8-hydroxy-2'-deoxyguanosine (8-OHdG) but not serum alkaline phosphatase activity. Interestingly, the diet induced significant increases of serum osteopontin and TGF-beta1, augumenting osteoclast-mediated bone resorption through the RANK/RANKL pathway [J. Clin. Invest. 112 (2003) 181]. TGF-beta1 showed a negative correlation with serum glutathione peroxidase (GPx) activity (r = -0.674, P < 0.003), but a positive correlation with the serum iron level (r = 0.836, P < 0.0001). Taken together, these results suggest for the first time that oxidative stress could be involved in the pathogenesis of metabolic bone diseases such as osteoporosis as demonstrated by analysis of the relationship between bone metabolism and oxidative stress.

Comparison between desferrioxamine and combined therapy with desferrioxamine and deferiprone in iron overloaded thalassaemia patients.

Desferrioxamine (DFX) alone (40-50 mg/kg/d s.c. over 8-12 h, five times weekly) was compared with combined DFX twice weekly and deferiprone (75 mg/kg/d) over 12 months in previously poorly chelated thalassaemia patients. Serum ferritin fell from 5506 +/- 635 microg/l (mean +/- SEM) to 3998 +/- 604 microg/l (P < 0.001; n = 14) in the DFX group and from 4153 +/- 517 microg/l to 2805 +/- 327 microg/l in the combined group (P < 0.01; n = 11). Deferiprone plus DFX produced a greater mean urine iron excretion (1.01 mg/kg/24 h) than iron intake from blood transfusion in each patient. Main side-effects were skin reactions (DFX alone), nausea and arthralgia (combined therapy). As chelation therapy, the combined protocol was as effective as DFX five times weekly.

Urine biochemical markers of early renal dysfunction are associated with iron overload in beta-thalassaemia.

Renal dysfunction in thalassemia patients can be attributed to chronic anemia, and iron overload as well as to desferioxamine (DFO) toxicity. We analyzed the urine of 91 well-maintained homozygous beta-thalassemia patients, with no evidence of renal disease, for early evidence of kidney dysfunction by means of electrophoresis and quantitative biochemical tests. Measurement of liver magnetic resonance imaging (MRI) T2 values and serum ferritin concentration was used to estimate iron overload. In 55 of the 91 patients, urine analysis indicated signs of tubular dysfunction. The urine concentration of albumin and beta 2-microglobulin, as well as the activity of N-acetyl-beta-D-glucosaminidase (NAG), correlated positively with serum ferritin concentration and liver iron deposition, as detected by MRI T2 values. This suggested that the cause of renal dysfunction in homozygous beta-thalassemia is iron overload. On the other hand, the same urine markers did not correlate with age, indicating that chronic anemia or desferrioxamine (DFO) treatment are not related to renal dysfunction in thalassemia.