A nanocellulose-based colorimetric assay kit for smartphone sensing of iron and iron-chelating deferoxamine drug in biofluids.

The current work describes the development of a "nanopaper-based analytical device (NAD)", through the embedding of curcumin in transparent bacterial cellulose (BC) nanopaper, as a colorimetric assay kit for monitoring of iron and deferoxamine (DFO) as iron-chelating drug in biological fluids such as serum blood, urine and saliva. The iron sensing strategy using the developed assay kit is based on the decrease of the absorbance/color intensity of curcumin-embedded in BC nanopaper (CEBC) in the presence of Fe(III), due to the formation of Fe(III)-curcumin complex. On the other hand, releasing of Fe(III) from Fe(III)-CEBC upon addition of DFO as an iron-chelating drug, due to the high affinity of this drug to Fe(III) in competition with curcumin, which leads to recovery of the decreased absorption/color intensity of Fe(III)-CEBC, is utilized for selective colorimetric monitoring of this drug. The absorption/color changes of the fabricated assay kit as output signal can be monitored by smartphone camera or by using a spectrophotometer. The results of our developed sensor agreed well with the results from a clinical reference method for determination of Fe(III) concentration in human serum blood samples, which revealed the clinical applicability of our developed assay kit. Taken together, regarding the advantageous features of the developed sensor as an easy-to-use, non-toxic, disposable, cost-effective and portable assay kit, along with those of smartphone-based sensing, it is anticipated that this sensing bioplatform, which we name lab-on-nanopaper, will find utility for sensitive, selective and easy diagnosis of iron-related diseases (iron deficiency and iron overload) and therapeutic drug monitoring (TDM) of iron-chelating drugs in clinical analysis as well.

An expedient reverse-phase high-performance chromatography (RP-HPLC) based method for high-throughput analysis of deferoxamine and ferrioxamine in urine.

The present study was planned to optimize and validate an expedient reverse-phase high chromatography (RP-HPLC) based protocol for the analysis of deferoxamine (DFO) and ferrioxamine (FO) in urinary execration of patients suffering ß-thalassemia major. The optimized RP-HPLC method was found to be linear over the wide range of DFO and FO concentration (1-90 µg/mL) with appreciable recovery rates (79.64-97.30%) of quality controls at improved detection and quantitation limits and acceptable inter and intraday variability. Real-time analysis of DFO and FO in the urine of thalassemic patients (male and female) at different intervals of Desferal®(Novartis Pharmaceuticals Corporation) injection revealed DFO and FO excretion at significantly (p < 0) different rates. The maximum concentrations of DFO (76.7 ± 3.06 µg/mL) and FO (74.2 ± 3.25 µg/mL) were found in urine samples, collected after 6 h of drug infusion while the minimum levels of DFO (1.10 ± 0.12 µg/mL) and FO (2.97 ± 0.13 µg/mL) were excreted by patients after 24 h. The present paper offers balanced conditions for an expedient, reliable and quick determination of DFO and FO in urine samples.

An extended-release formulation of desferrioxamine for subcutaneous administration.

Desferrioxamine mesylate (DFO) remains the first line iron chelating agent. Since it has a short half-life and poor absorption through the gastrointestinal tract, DFO must be administered parenterally, usually by daily subcutaneous infusion administered over 8-12 h. The objective of this paper was the development of multivesicular liposome (depofoam) for the extended-release of DFO and study of iron excretion efficiency compared to the free form of DFO. Depofoam particles were characterized by their morphology, particle size, capture volume, and in vitro release. Also, in vivo activity of this formulation in iron overload rats was studied. The in vitro studies in 0.9% sodium chloride at 37 degrees C showed that the multivesicular liposomes released DFO slowly over several days without a rapid initial release, and 57% of DFO was released in 9 days. Administration of a single dose of 100 mg/kg of an optimized Depo-DFO formulation in an iron overload rats, as a single bolus subcutaneous injection, led to significant elevation of urinary iron excretion at the first day that were maintained at levels of more than 110 microg/kg for 3 days. Administration of the unencapsulated DFO at the same dose resulted in elevation of urinary iron excretion in the first day (approximately 73% amount of iron excretion by Depo-DFO) followed by a quick decline to base line levels in the second day. The total urinary iron excreted by Depo-DFO is 3-times greater than that elicited by DFO. In conclusion, Depo-DFO appears to have potential usefulness as an extended-release formulation of DFO.

A high-performance liquid chromatographic method for the measurement of deferoxamine in body fluids.

A high-performance liquid chromatography method for the analysis of deferoxamine (DFO) in 100 microliters of serum or plasma is described. The procedure involves the addition of the internal standard ciprofloxacin to the sample, followed by ultrafiltration to remove protein. The ultrafiltrate is then directly injected into the chromatography system. Separation is achieved using a reverse-phase mu Bondapak C18 column and a ternary solvent system (sodium phosphate:acetonitrile:methanol) running at 2.0 ml/min. Assay time is 10 min, and chromatograms show no interference from coadministered drugs during this period of time. Coefficients of variation were found to be less than 5%, and analytical recovery of DFO was 85%. Validation experiments in an experimental dog model and in patients with iron overload demonstrate that the method is appropriate for studying the pharmacokinetics of DFO in thalassemic patients receiving drug for the treatment of chronic iron overload.

Pharmacokinetics and renal elimination of desferrioxamine and ferrioxamine in healthy subjects and patients with haemochromatosis.

1 Desferrioxamine mesylate (DM) (10 mg kg-1 = 15.24 mumol kg-1) was given by intramuscular injection to five healthy subjects and to six patients with haemochromatosis, after informed consent. 2 Desferrioxamine (DFA), ferrioxamine (FeA), aluminoxamine (AlA), aluminium (Al) and iron (Fe) were measured in plasma, before and 10, 20, 30, 60 min and 2, 4, 6, 8, 12 h after DM injection and in urine collected over a 6 h period the day before and the day of administration. 3 The predominant form in plasma from control subjects was DFA whereas FeA predominated in plasma from patients. In controls, rapid and slow phases of decline in plasma DFA concentrations were found, with half-lives of 1.0 h and 6.1 h, respectively. In the patients, only a single phase of decline was observed, with a half-life of 5.6 h. Total clearances of DFA were 296 ml h-1 kg-1 in controls and 239 ml h-1 kg-1 in patients. 4 The amount of FeA eliminated in urine during 6 h was significantly lower in controls (8.0 +/- 4.6 mumol) than in patients (129.2 +/- 40.0 mumol), with respective renal clearances estimated over 6 h of 516 ml h-1 kg-1 and 1,716 ml h-1 kg-1. DFA elimination was similar in both groups and its renal clearance estimated over 6 h was 91 ml h-1 kg-1 in controls and 85 ml h-1 kg-1 in patients. 5 Since there was no overlap in the 1 h DFA/FeA plasma ratio between controls and patients, this might be useful as an index of iron overload.

Quantification of desferrioxamine in blood plasma by inductively coupled plasma atomic emission spectrometry.

A sensitive method, inductively coupled plasma atomic emission spectroscopy, is used to measure desferrioxamine in blood plasma. The desferrioxamine is transformed into its iron chelate, ferrioxamine, which is extracted into benzyl alcohol, then re-extracted into HCl (0.5 mol/L), which is used as the sample for the spectroscopy. For a 0.5-mL plasma sample, the detection limit (1 microgram/mL) suffices for following the concentration of desferrioxamine in plasma after its subcutaneous or intramuscular injection (40 mg per kg of body weight). Neither blood pigments nor trace metals interfere.

Management of acute iron poisoning.

Acute iron poisoning is most common in children below the age of 5 years. While there is no doubt that it may be fatal, recent surveys show that death occurs in only a very small percentage of cases and that iron salts are responsible for a small minority of fatalities due to overdosage with drugs. Similarly, the proportion of severe cases seems to have fallen over the last thirty years, possibly due to earlier and more aggressive treatment but more probably due to an increase in the number of minor exposures reported. Iron salts are directly toxic to the gastrointestinal tract causing vomiting, diarrhoea, abdominal pain and occasionally significant blood loss. They also cause metabolic acidosis by interfering with intermediary metabolism and producing shock and reduced tissue perfusion. The clinical course of acute iron poisoning is divided into 4 phases. Features of acute gastrointestinal irritation dominate the period up to 6 hours after ingestion and most patients do not develop other features or progress beyond this stage. Rarely, blood loss may be sufficient to cause hypotension. Severe poisoning is characterised by impairment of consciousness, convulsions and metabolic acidosis. The second phase, 6 to 12 hours after ingestion, is one of remission of features. Phase 3 comprises the period 12 to 48 hours from ingestion and is reached only by a small minority of patients. Recurrence or development of shock, and metabolic acidosis are usual and renal failure and features of extensive hepatocellular necrosis may develop. The last (fourth) phase, 2 to 6 weeks after ingestion, is only likely to develop in young children and is characterised by recurrence of vomiting due to gastric or duodenal stenosis caused by healing of iron-induced mucosal ulcers. Acute iron poisoning in humans has not been adequately studied and is unlikely to be so now because of the infrequent and sporadic occurrence of cases. The evidence for many conventional aspects of management is therefore unsatisfactory. Assessment of severity of poisoning is an essential prerequisite to optimum management but is difficult. The amount of elemental iron ingested is unacceptable since it is seldom known with accuracy and absorption is unpredictable because of vomiting and diarrhoea. The commonly encountered clinical features are also unreliable although it is generally accepted that coma, shock and metabolic acidosis indicate severe poisoning.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid determination of iron in urine, in the presence of deferoxamine, by inductively coupled plasma emission spectrometry.

Using a spectrometer with an argon plasma source coupled to a high-frequency magnetic field, we developed a direct method for determining iron in urine of patients being treated with deferoxamine. The detection limit for iron was 75 nmol/L; added iron was satisfactorily recovered; and we observed no interference from deferoxamine at its most commonly used concentrations. Values for between-run and within-run precision (CV) was less than 5%. Correlation of results with those obtained with a colorimetric method involving bathophenanthroline was good (r = 0.96).

Determination of desferoxamine and a major metabolite by high-performance liquid chromatography. Application to the treatment of aluminium-related disorders.

A high-performance liquid chromatography method is described that permits separation and quantification of desferoxamine, a major metabolite, the iron(III) and the aluminum(III) chelates of desferoxamine. This method now facilitates pharmacokinetic studies on desferoxamine and derivatives designed to study side-effects and metabolite patterns in patients undergoing treatment.

Iron overload disorders: natural history, pathogenesis, diagnosis, and therapy.

Hemochromatosis is a syndrome which, when fully expressed, is manifested by melanoderma , diabetes mellitus, and liver cirrhosis, with iron overload involving parenchymal and reticuloendothelial cells in many organ systems. This clinical presentation may arise as a consequence of either hereditary or acquired abnormalities of iron overload, although the mechanisms are quite different. In hereditary hemochromatosis (also known as primary, or idiopathic, hemochromatosis), increased intestinal iron absorption leads to excessive accumulations of iron, throughout the body, particularly in parenchymal cells. In secondary forms of iron overload including transfusional hemosiderosis, alcoholic cirrhosis, thalassemia, sideroblastic anemia, and porphyria cutanea tarda, iron accumulates in the reticuloendothelial system initially, but with increasing amounts of total body iron, excessive iron deposits eventually accumulate in parenchymal cells throughout the body producing a picture indistinguishable from hereditary hemochromatosis. In this article, the course, prognosis, and therapy of iron overload will be reviewed in detail. Clinical and experimental data concerning the pathogenesis of the different forms of iron overload will be examined critically. In particular, information relating to possible abnormalities of reticuloendothelial function, intestinal mucosal iron transport, and alterations in serum and tissue isoferritin patterns in hereditary hemochromatosis will be analyzed, and possible directions for future research will be suggested. The mode of inheritance and linkage with the major histocompatibility (HLA) complex will be discussed. Theories on the pathogenesis of tissue damage by excess iron will be evaluated. Methods for measuring the extent of iron overload in clinical practice will be described, including measurements of serum iron, serum ferritin, iron absorption, cobalt excretion, desferrioxamine excretion, liver biopsy and tissue iron determinations, and HLA typing. Finally, unresolved problems in the understanding of the disease process, diagnosis, and therapy will be delineated.

A preliminary study of iron absorption by whole body counting and correlation with DFO excretion.

A preliminary study of iron absorption by whole body counting was carried on a group of 16 women. The cases included 8 patients suffering from iron deficiency anaemia and various infections as well as 8 healthy controls. High iron absorption is associated with iron deficiency, these changes being more marked in iron deficient controls than in those with infection or malignancy. In iron deficient controls results of whole body counting correlate very well with other haematological investigations.