Curcumin as Treatment for Bladder Cancer: A Preclinical Study of Cyclodextrin-Curcumin Complex and BCG as Intravesical Treatment in an Orthotopic Bladder Cancer Rat Model.

OBJECTIVE: To evaluate the antitumor effect of cyclodextrin-curcumin complex (CDC) on human and rat urothelial carcinoma cells in vitro and to evaluate the effect of intravesical instillations of CDC, BCG, and the combination in vivo in the AY-F344 orthotopic bladder cancer rat model. Curcumin has anticarcinogenic activity on urothelial carcinoma and is therefore under investigation for the treatment of non-muscle invasive bladder cancer. Curcumin and BCG share immunomodulating pathways against urothelial carcinoma. METHODS: Curcumin was complexed with cyclodextrin to improve solubility. Four human urothelial carcinoma cell lines and the AY-27 rat cell line were exposed to various concentrations of CDC in vitro. For the in vivo experiment, the AY-27 orthotopic bladder cancer F344 rat model was used. Rats were treated with consecutive intravesical instillations of CDC, BCG, the combination of CDC+BCG, or NaCl as control. RESULTS: CDC showed a dose-dependent antiproliferative effect on all human urothelial carcinoma cell lines tested and the rat AY-27 urothelial carcinoma cell line. Moreover, intravesical treatment with CDC and CDC+BCG results in a lower percentage of tumors (60% and 68%, respectively) compared to BCG (75%) or control (85%). This difference with placebo was not statistically significant (p=0.078 and 0.199, respectively). However, tumors present in the placebo and BCG-treated rats were generally of higher stage. CONCLUSIONS: Cyclodextrin-curcumin complex showed an antiproliferative effect on human and rat urothelial carcinoma cell lines in vitro. In the aggressive orthotopic bladder cancer rat model, we observed a promising effect of CDC treatment and CDC in combination with BCG.

Cyclodextrin curcumin formulation improves outcome in a preclinical pig model of marginal kidney transplantation.

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Heme oxygenase 1 gene polymorphisms and outcome of renal transplantation.

Heme oxygenase isoenzyme HO-1 has been linked to several cytoprotective functions with a potentially beneficial role in transplantation. In the present study, the effect of genetic variation in HO-1 on renal allograft outcome was investigated. Six hundred and eighty patients subject to renal transplantation in a single transplant unit and their cadaveric kidney donors were included in this study. Four single-nucleotide polymorphisms and one microsatellite marker in the HO-1 gene region were analysed. Some statistically nominally significant associations were observed in preliminary analyses between polymorphisms studied and clinical outcomes, but after correction for multiple comparisons none remained significant. Our data suggest that the HO-1 gene polymorphisms studied have no significant role on outcome of kidney transplantation in the Finnish population.

Redox-active iron released during machine perfusion predicts viability of ischemically injured deceased donor kidneys.

Redox-active iron, catalyzing the generation of reactive oxygen species, has been implicated in experimental renal ischemia-reperfusion injury. However, in clinical transplantation, it is unknown whether redox-active iron is involved in the pathophysiology of ischemic injury of non-heart-beating (NHB) donor kidneys. We measured redox-active iron concentrations in perfusate samples of 231 deceased donor kidneys that were preserved by machine pulsatile perfusion at our institution between May 1998 and November 2002 using the bleomycin detectable iron assay. During machine pulsatile perfusion, redox-active iron was released into the preservation solution. Ischemically injured NHB donor kidneys had significantly higher perfusate redox-active iron concentrations than heart-beating (HB) donor kidneys that were not subjected to warm ischemia (3.9 +/- 1.1 vs. 2.8 +/- 1.0 micromol/L, p = 0.001). Moreover, redox-active iron concentration was an independent predictor of post-transplant graft viability (odds ratio 1.68, p = 0.01) and added predictive value to currently available donor and graft characteristics. This was particularly evident in uncontrolled NHB donor kidneys for which there is the greatest uncertainty about transplant outcomes. Therefore, perfusate redox-active iron concentration shows promise as a novel viability marker of NHB donor kidneys.

A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance.

BACKGROUND AND OBJECTIVES: The increasing demand for intravenous immunoglobulin (IVIG) necessitates the development of improved plasma fractionation methods, providing higher immunoglobulin G (IgG) recovery. Here, we describe a new IVIG production process resulting in a high yield of IgG and effective reduction of physico-chemically resistant viruses. MATERIALS AND METHODS: IgG was purified from Cohn fraction II+III by caprylic acid treatment, polyethylene glycol precipitation, anion-exchange chromatography, nanofiltration and ultrafiltration. Stability of the purified IgG was studied in different formulations. Virus reduction was studied with two viruses: bovine viral diarrhoea virus, assessed by an infectivity assay; and human parvovirus B19, assessed by polymerase chain reaction. RESULTS: The combination of caprylic acid treatment with polyethylene glycol precipitation and a single anion-exchange chromatography yielded polymer-free, pure IgG. The purified IgG could be filtered through a small pore-size virus filter (Millipore V-NFP) with high throughput and excellent yield. The formulated product was stable as a 100 g/l IgG solution. Bovine viral diarrhoea virus was effectively inactivated by the caprylic acid treatment, and parvovirus B19 was effectively removed in the polyethylene glycol precipitation and nanofiltration stages, the total reduction of parvovirus being approximately 14 log10. CONCLUSIONS: The new process gives pure and stable IgG solution with an average yield of 4.8 g of IgG per kg of recovered plasma and has a very high capacity to remove even physico-chemically resistant viruses.

Viral safety of Nanogam, a new 15 nm-filtered liquid immunoglobulin product.

BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).

Aneurysmal bone cyst of the index sesamoid.

Sesamoid bone disorders are rare and usually are a result of trauma or degenerative causes. Tumors of the sesamoid bones of the hand are encountered less frequently. We report a case of an aneurysmal bone cyst of the radial index sesamoid of the hand. The tumor was treated successfully by sesamoid bone resection.

Development of a highly purified multicomponent leukocyte IFN-alpha product.

A purification process was developed to obtain a human interferon- alpha (IFN-alpha) product that contains all major IFN-alpha subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-alpha in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 x 10(8) IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-alpha 1, IFN-alpha 2, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 17, and IFN-alpha 21. The subtypes IFN-alpha 4 and IFN-alpha 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilization of the pure IFN-alpha solution with albumin and Tween 80 was compared. In virus filtration, a better yield and higher filtration capacity were obtained with Tween. The addition of albumin resulted in the formation of IFN-albumin aggregates. During long-term storage, IFN-alpha was stable in both solutions for 2 years at 2-8 degrees C. The new method makes it possible to extensively purify all major IFN-alpha subtypes and obtain a virus-safe and stable product with a constant subtype composition.

Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice.

We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N(1),N(11)-diethylnorspermine. In tracer experiments with [(14)C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.

HSV-tk gene therapy for human renal cell carcinoma in nude mice.

We have treated Caki-2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Both stably transduced Caki-2 tumors, generated using retrovirus-mediated ex vivo HSV-tk gene transfer and direct intratumoral adenovirus-mediated HSV-tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro- and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki-2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild-type tumors in vivo with adeno-LacZ was 22+/-14%. Significant tumor regression was achieved with direct intratumoral adeno-HSV-tk transduction followed by intraperitoneal ganciclovir (GCV) (P<.001). Also, the treatment of stably transduced Caki-2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV-tk group as compared to the LacZ group (P<.009). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV-tk and GCV. Also, significant prolongation in survival was achieved with adeno-HSV-tk- and GCV-treated mice as compared to the controls. It is concluded that adeno-HSV-tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo.

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.

Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase.

We recently generated a transgenic mouse line with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase. Phenotypic changes in these animals included permanent loss of hair at the age of 3 wk. We have now further explored development of hair loss during early postnatal life. The first hair cycle appeared to be completed normally in the transgenic animals. At postnatal day 15, although macroscopically indistinguishable from their syngenic littermates, the transgenic animals already showed microscopically signs of hair follicle degeneration. Wild-type mice started their second anagen phase at day 27, whereas the transgenic animals did not display functional hair follicles at that time. Hair follicles were replaced by dermal cysts and epidermal utriculi. Analysis of skin polyamines revealed that the transgenic animals continuously overaccumulated putrescine. The view that an overaccumulation of putrescine was related to the disturbed hair follicle development was strengthened by the finding that doubly transgenic mice overexpressing, both spermidine/spermine N1-acetyltransferase and ornithine decarboxylase and with extremely high levels of putrescine in the skin, showed distinctly more severe skin changes compared with the singly transgenic animals. Interest ingly, in spite of their hairless phenotype, the spermidine/spermine N1-acetyltransferase transgenic mice, were significantly more resistant to the development of papillomas in response to the two-stage skin carcinogenesis. Analysis of skin polyamines indicated that the syngenic mice tripled their spermidine content when exposed to promotion, whereas the transgenic animals showed only modest changes. These results suggest that putrescine plays a pivotal part in normal hair follicle development.

Non-transferrin-bound iron during allogeneic stem cell transplantation.

Hydroxyl radical formation catalysed by non-transferrin-bound iron (NTBI) might contribute to transplantation-related complications. The occurrence of NTBI in 10 adult allogeneic stem cell transplantation (SCT) patients was followed for 20 d. The transferrin saturation reached 99% on d -4 and remained > 80% thereafter. NTBI, measured as bleomycin-detectable iron, was detected for 6-18 d in all patients with a peak on d -4. High transferrin saturation levels were associated with the appearance of NTBI with a threshold at 80% saturation. Prevention of the potential deleterious effects of NTBI might reduce transplantation-related morbidity.

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth.

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.

Prognostic value of hyaluronan expression in non-small-cell lung cancer: Increased stromal expression indicates unfavorable outcome in patients with adenocarcinoma.

The prognostic value of hyaluronan (HA) was analyzed in a large number of patients (n = 261) with non-small-cell lung cancer (NSCLC) by staining archived tumor samples with a biotinylated HA-specific probe. The level of HA in the tumor cells and surrounding stroma was scored and compared with parallel CD44 stainings, clinicopathological factors and survival data. Adenocarcinomas were characterized by a low percentage of HA-positive cells with low staining intensity compared with squamous-cell and large-cell/anaplastic carcinomas. The HA signal in the peri-tumoral stroma was often higher than that in the uninvolved stroma in all subgroups of NSCLC. CD44 and HA associated with the cancer cells showed a strong positive correlation with each other. In the whole tumor material, dominated by squamous-cell carcinomas (n = 168), recurrences were more often found in cases showing a low percentage of cancer cell-associated HA. However, within the adenocarcinoma subgroup (n = 68), a high percentage of cell-associated HA was correlated with poor tumor differentiation. Also specific for the adenocarcinoma subgroup was the increased number of recurrences in cases with a strong stromal HA signal. In survival analysis of the whole material (n = 189), a low percentage of HA-positive cancer cells was associated with a shortened disease-free survival (DFS) together with stage and tumor type. However, in the subgroup of patients with adenocarcinoma (n = 49), a strong stromal signal for HA predicted poor DFS. The level of HA in the stroma of adenocarcinomas retained its prognostic value in Cox's multivariate analysis. These results indicate that the frequency and intensity of HA has a significant prognostic value in NSCLC, particularly when the histological subtypes are analyzed as separate entities.

Spermine deficiency resulting from targeted disruption of the spermine synthase gene in embryonic stem cells leads to enhanced sensitivity to antiproliferative drugs.

Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.

Age matters: collagen birefringence of superficial articular cartilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading.

OBJECTIVE: To compare responses of the collagen network and glycosaminoglycans (GAGs) of articular cartilage to physiological type of joint loading in young growing and adult mature guinea-pigs. DESIGN: 10- and 44-week-old guinea-pigs were accustomed to treadmill running for 3 weeks. Thereafter the animals ran 2500 m/day, 5 days a week, for 15 weeks. Articular cartilage specimens from knee joints were collected at 28 and 62 weeks. Osteoarthritis (OA) prevalence and severity was evaluated by aid of light microscopy. The degree of collagen fibril network organization and content was analyzed with quantitative polarized light microscopy. The local concentration of GAGs was determined from cartilage sections with digital densitometry after safranin-O staining. RESULTS: In the young guinea-pigs, running increased up to 24% the optical retardation of polarized light by collagen in the superficial articular cartilage of femur, indicating either a higher degree of fibril assembly and organization or increased amount of collagen, or both. In contrast, in the adult mature animals the optical retardation decreased almost 50% after joint loading (P< 0.01-0.001). Running did not increase cartilage fibrillation. Significant changes in GAG content of cartilage were not found either in the young or adult mature runners. CONCLUSIONS: Increased birefringence of the superficial articular cartilage after joint loading in young guinea-pigs can be interpreted to be a sign of improved and decreased birefringence in older animals a sign of worsened property of the collagen network. It can be suggested therefore that joint loading strengthened the collagen network in the young runners. It can be hypothesized further that with time the inferior property of the collagen network predisposes the older runners to earlier OA than in controls.

Catalytically active iron and bacterial growth in serum of haemodialysis patients after i.v. iron-saccharate administration.

BACKGROUND: I.v. iron is commonly administered to haemodialysis patients suffering from anaemia to improve their response to erythropoietin therapy. It has been unclear whether routinely used doses of i.v. iron preparations could result in iron release into plasma in amounts exceeding the iron binding capacity of transferrin. Here, we have studied the effect of 100 mg of iron saccharate given as an i.v. injection on transferrin saturation and the appearance of potentially harmful catalytically active iron. METHODS: We followed serum iron, transferrin and transferrin-saturation before and 5-210 min after administration of iron saccharate in 12 patients on chronic haemodialysis due to end-stage renal disease. We measured catalytically active iron by the bleomycin-detectable iron (BDI) assay and transferrin iron forms by urea gel electrophoresis, and studied iron-dependent growth of Staphylococcus epidermidis inoculated into the serum samples in vitro. RESULTS: The iron saccharate injection resulted in full transferrin saturation and appearance of BDI in the serum in seven out of the 12 patients. BDI appeared more often in patients with a low serum transferrin concentration, but it was not possible to identify patients at risk based on serum transferrin or ferritin level before i.v. iron. The average transferrin saturation and BDI level increased until the end of the follow-up time of 3.5 h. The appearance of BDI resulted in loss of the ability of patient serum to resist the growth of S. epidermidis, which was restored by adding iron-free apotransferrin to the serum. Iron saccharate, added to serum in vitro, released only little iron and promoted only slow bacterial growth, but caused falsely high transferrin saturation by one routinely used serum iron assay. CONCLUSIONS: The results indicate that 100 mg of iron saccharate often leads to transferrin oversaturation and the presence of catalytically active iron within 3.5 h after i.v. injection. As catalytically active iron is potentially toxic and may promote bacterial growth, it may be recommendable to use dosage regimens for i.v. iron that would not cause transferrin oversaturation.