Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

Biodistribution, Clearance And Morphological Alterations Of Intravenously Administered Iron Oxide Nanoparticles In Male Wistar Rats.

INTRODUCTION: Nanoparticles are used worldwide because of their unique properties, with large-scale application in various fields, such as medicine, cosmetics and industries. In view of their widespread use, the potential adverse effects of nanoparticles have become a significant cause for concern, in terms of not only human health and safety but also the environment. The present investigation focused on establishing the bioaccumulation patterns and ultrastructural changes induced by retained iron oxide nanoparticles (IONPs) in various target organs of rats. METHODS: Twenty-four male Wistar rats were randomly divided into four groups. Experimental animals were intravenously administered different doses of IONPs (7.5 mg/kg, 15 mg/kg and 30 mg/kg) once in a week for 4 weeks. Urine and feces samples were collected on a daily basis to assess nanoparticle clearance and analyzed via atomic absorption spectroscopy (AAS). At the end of the experiment, rats were euthanized and different organs, including spleen, liver, kidney, lung, heart, testis and brain, were dissected. Bioaccumulation of iron in organs and ultrastructural changes induced by IONPs were determined. RESULTS: The maximal concentration of iron was detected in spleen and minimal concentration in the brain. The level of iron accumulation in organs was as follows: spleen>blood>liver>kidney>lung>heart>testis>brain. The excretion profile in urine revealed maximum excretion on the day following administration that was maintained until day 28, whereas the iron content in feces remained high during the first three days after injection. A similar pattern was observed throughout the duration of the experiment. Ultrastructural alterations were detected in spleen, kidney, lung, heart, testis, brain and liver, indicative of cellular damage induced by accumulating nanoparticles in these organs. CONCLUSION: Intravenous administration of IONPs results in ultrastructural changes and dose-dependent bioaccumulation in different organs of rats.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Facile preparation and characterization of new green emitting carbon dots for sensitive and selective off/on detection of Fe3+ ion and ascorbic acid in water and urine samples and intracellular imaging in living cells.

Carbon dots (CDs) have gained great attention as multifunctional materials because of their interesting properties and general applicability. However, there are some reports for the preparation of highly luminescent green-emitting CDs (G-CDs), although these reports seem not to be extensible. Herein, new G-CDs (quantum yield: 27.2%) were synthesized from a facile hydrothermal treatment of p-aminosalicylic acid and ethylene glycol dimethacrylate as both carbon and nitrogen source and cross-linking agent, respectively. The chemical composition and optical properties of the as-prepared G-CDs were successfully investigated using transmission electron microscopy, atomic force microscopy, dynamic light scattering, X-ray diffraction, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and fluorescence and UV-vis spectroscopies. Interestingly, the fluorescence intensity of G-CDs was selectivity quenched by Fe3+ in the range of 0.05-10.0 µmol L-1, with a detection limit of 13.7 nmol L-1. Meanwhile, ascorbic acid found to reduce Fe3+ to Fe2+, thereby causing restoration of the fluorescence of G-CDs. The detection limit for ascorbic acid detection was estimated as 82.0 nmol L-1 over a linear range from 0.2 to 11.0 µmol L-1. Furthermore, the designed sensing platform was successfully utilized to the detection of Fe3+ and ascorbic acid in water and urine samples and to intracellular imaging without surface modification.

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.

Tandem measurements of iron and creatinine by cross injection analysis with application to urine from thalassemic patients.

This work presents development of a method for the dual determination of Fe(III) and creatinine using cross injection analysis (CIA). Two CIA platforms connected in series accommodated sample and reagents plugs aspirated via y-direction channels while water was pumped through the x-direction channel toward a flow-through cell of a diode array UV-vis. detector. Iron was detected from the colorimetric reaction between Fe(II) and 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-(3-sulfopropyl)amino) aniline (5-Br-PSAA), with prior reduction of Fe(III) to Fe(II) by ascorbic acid. The Jaffe's reaction was employed for the detection of creatinine. Under the optimal conditions, good linearity ranges were achieved for iron in the range 0.5 to 7 mg L(-1) and creatinine in the range 50 to 800 mg L(-1). The CIA system was applied to spot urine samples from thalassemic patients undergoing iron chelation therapy, and was successfully validated with ICP-OES and batchwise Jaffe's method. Normalization of urinary iron excretion with creatinine is useful for correcting the iron concentration between urine samples due to variation of the collected urine volume.

Novel DFO-functionalized mesoporous silica for iron sensing. Part 2. Experimental detection of free iron concentration (pFe) in urine samples.

Successful in vivo chelation treatment of iron(iii) overload pathologies requires that a significant fraction of the administered drug actually chelates the toxic metal. Increased mobilization of the iron(iii) in experiments on animals or humans, most often evaluated from urinary output, is usually used as an assessment tool for chelation therapy. Alternatively, the efficiency of a drug is estimated by calculating the complexing ability of a chelating agent towards Fe(iii). The latter is calculated by the pFe value, defined as the negative logarithm of the concentration of the free metal ion in a solution containing 10 µM total ligand and 1 µM total metal at a physiological pH of 7.4. In theory, pFe has to be calculated taking into account all the complexation equilibria involving the metal and the possible ligands. Nevertheless, complexation reactions in complex systems such as serum and urine may hardly be accurately modelled by computer software. The experimental determination of the bioavailable fraction of iron(iii) in biological fluids would therefore be of the utmost relevance in the clinical practice. The efficiency of the therapy could be more easily estimated as well as the course of overload pathologies. In this context, the aim of the present work was the development of a sensor to assess the free iron directly in biological fluids (urine) of patients under treatment with chelating agents. In the proposed device (DFO-MS), the strong iron chelator deferoxamine (DFO) is immobilized on the MCM-41 mesoporous silica. The characterization of the iron(iii) sorption on DFO-MS was undertaken, firstly in 0.1 M KNO3, then directly in urine samples, in order to identify the sorption mechanism. The stoichiometry of the reaction in the solid phase was found to be: with an exchange constant (average value) of log ßex = 40(1). The application of DFO-MS to assess pFe in SPU (Simulating Pathology Urine) samples was also considered. The results obtained were very promising for a future validation and subsequent application of the sensor in samples of patients undergoing chelation therapy.

Role of digoxin-like immunoreactive substance in the pathogenesis of transient tachypnea of newborn.

BACKGROUND: Transient tachypnea of newborn (TTN) is usually observed in term or near-term infants. It constitutes an important part of the respiratory distress cases observed in the neonatal intensive care unit (NICU). AIM: This paper examines the effects of digoxin-like immunoreactive substance (DLIS) on fluid and ion balance, hemodynamic and echocardiographic parameters of neonates with TTN. METHODS: Plasma DLIS, Na(+), K(+), urea, creatinine, serum and urine osmolarity, urine FeNa(+), 24-hour urine output, echocardiographic investigation and mean blood pressure, and clinical parameters of disease severity were recorded in TTN group and compared with control on the 1st and 7th days of their lives. RESULTS: Plasma DLIS levels were statistically higher in TTN group (0.66 ± 0.37 ng/mL) compared to control group (0.24 ± 0.20 ng/mL) both on the 1st day (P < 0.01) and the 7th day (P < 0.05). For TTN group, significant correlation was found between plasma DLIS levels and maximum respiratory rate, duration of tachypnea, and length of hospitalization on the 1st day. Plasma DLIS levels were correlated negatively with serum osmolarity levels. Plasma DLIS levels were positively correlated with urine output, urinary FeNa(+) levels, cardiac output, left ventricles end diastolic diameters, and right ventricles end diastolic diameters. CONCLUSIONS: Increased DLIS levels were correlated with disease severity in cases with TTN. This increase may be a primary or secondary event in the disease progress. It may help reduce the fluid overload due to already disturbed cardiac functions in patients by increasing urine output and natriuresis; however it may also contribute to disease pathogenesis, by inhibiting alveolar Na(+)-K(+)-ATPase which further decreases fetal alveolar fluid resorption.

High levels of oxidative stress in rats infected with Blastocystis hominis.

OBJECTIVE: Numerous studies have revealed the presence of oxidative stress in parasitic infections. However, such studies were lacking in the Malaysian population. Previously, we have provided evidence that oxidative stress is elevated in Malaysians infected with intestinal parasites. Stool examinations revealed that about 47.5% of them were infected with the polymorphic protozoa, Blastocystis hominis. However, they were found to have mixed infection with other intestinal parasites. METHODOLOGY: Therefore, in order to investigate the role of B. hominis alone in affecting oxidative stress status, here we compared the levels of oxidative stress biomarkers in urine and blood samples between uninfected and B. hominis-infected rats. RESULTS: Infected rats exhibited elevated levels of oxidative indices namely advanced oxidative protein products (AOPP), hydrogen peroxide (H2O2) and lipid hydroperoxide (LHP) indicating that their overall oxidative damage level was higher. Ferric reducing antioxidant power (FRAP) was elevated at the initial stage of infection but decreased significantly during the last week of study duration suggesting that the antioxidant status of the host may be overwhelmed by oxidative damage. CONCLUSION: To date, this is the first comprehensive in vivo study to provide evidence for Blastocystis infection to correlate with significant oxidative burst leading to oxidative stress.

Pulmonary toxicity and kinetic study of Cy5.5-conjugated superparamagnetic iron oxide nanoparticles by optical imaging.

Recent advances in the development of nanotechnology and devices now make it possible to accurately deliver drugs or genes to the lung. Magnetic nanoparticles can be used as contrast agents, thermal therapy for cancer, and be made to concentrate to target sites through an external magnetic field. However, these advantages may also become problematic when taking into account safety and toxicological factors. This study demonstrated the pulmonary toxicity and kinetic profile of anti-biofouling polymer coated, Cy5.5-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) by optical imaging. Negatively charged, 36 nm-sized, Cy5.5-conjugated TCL-SPION was prepared for optical imaging probe. Cy5.5-conjugated TCL-SPION was intratracheally instilled into the lung by a non-surgical method. Cy5.5-conjugated TCL-SPION slightly induced pulmonary inflammation. The instilled nanoparticles were distributed mainly in the lung and excreted in the urine via glomerular filtration. Urinary excretion was peaked at 3 h after instillation. No toxicity was found under the concentration of 1.8 mg/kg and the half-lives of nanoparticles in the lung and urine were estimated to be about 14.4+/-0.54 h and 24.7+/-1.02 h, respectively. Although further studies are required, our results showed that Cy5.5-conjugated TCL-SPION can be a good candidate for use in pulmonary delivery vehicles and diagnostic probes.

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.

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)

Acute renal failure and glucosuria induced by ferric nitrilotriacetate in rats.

Nitrilotriacetate (NTA), an effective metal-chelating agent, has been used as a substitute for polyphosphates in household laundry detergents. Nephrotoxicity and renal tumorigenicity have been reported in experimental animals that received high doses of NTA po for 4 weeks to 2 years. Since NTA exists in water as a variety of NTA-metal complexes, it was important to investigate the biological effects of NTA in a complexed form. In this study, acute and subchronic toxicity of a ferric iron chelate of NTA (Fe-NTA) was investigated in rats. When Fe-NTA was given ip, acute tubular necrosis and renal failure occurred following a single injection of 15 mg iron/kg. Repeated injections of sublethal doses produced degeneration and necrosis of the proximal tubular epithelium and was associated with polyuria, glucosuria, aminoaciduria, and azotemia. After 9 days of treatment, regeneration of the tubular epithelium with atypical cells was observed. Except for a parenchymal iron deposit, no marked changes were observed in other organs. None of these effects were observed in animals given noncomplexed NTA. In conclusion, the toxicity observed following high doses of NTA given po may be the result of an absorbed metal-NTA chelate.

Factors affecting the absorption of iron from Fe(III)EDTA.

1. The modification of iron absorption from Fe(III)EDTA by agents known to promote or inhibit absorption was examined in 101 volunteer multiparous Indian women. Fe absorption from Fe(III)EDTA was compared with absorption of intrinsic food Fe in a further twenty-eight subjects. Finally the urinary excretion of radio-Fe after oral administration of 59Fe(III)EDTA was studied in twenty-four subjects and evidence of intraluminal exchange of Fe was examined. 2. Fe absorption from maize porridge fortified with Fe(III)EDTA was more than twice that from porridge fortified with FeSO4 . 7H2O. 3. Although bran decreased Fe absorption from FeSO4 . 7H2O approximately 11-fold, it had no significant effect on Fe absorption from Fe(III)EDTA. Nevertheless tea, which is a more potent inhibitor of Fe absorption, decreased absorption from Fe(III)EDTA 7-fold. 4. Fe absorption from Fe(III)EDTA given in water was only increased 40% by addition of 3 mol ascorbic acid/mol Fe but by 7-fold when the relative proportions were increased to 6:1. This enhancing effect was blunted when the Fe(III)EDTA was given with maize porridge. In these circumstances, an ascorbate:iron value of 3:1 (which doubles absorption from FeSO4 . 7H2O) produced no significant increase in Fe absorption, while a value of 6:1 produced only a 2 . 5-fold increase. 5. Fe absorption from Fe(III)EDTA was not altered by addition of maize porridge unless ascorbic acid was present. 6. Less than 1% of 59Fe administered as 59Fe(III)EDTA was excreted in the urine and there was no inverse relationship between Fe absorption and the amounts excreted (r 0 . 58, P less than 0 . 05). 7. Isotope exchange between 59Fe(III)EDTA and 59FeSO4 . 7H2O was demonstrated by finding a similar relative value for the two isotopes in urine and erythrocytes when the two labelled compounds were given together orally. This finding was confirmed by in vitro studies, which showed enhanced 59Fe solubilization from 59FeSO4 . 7H2O in maize porridge when unlabelled Fe(III)EDTA was added. 8. Although Fe absorption from Fe(III)EDTA was marginally higher it appeared to form a common pool with intrinsic food iron in most studies. It is postulated that the mechanism whereby Fe(III)EDTA forms a common pool with intrinsic food Fe differs from that occurring with simple Fe salts. When Fe is present in the chelated form it remains in solution and is relatively well absorbed because it is protected from inhibitory ligands. Simple Fe salts, however, are not similarly protected and are absorbed as poorly as the intrinsic food Fe. 9. It is concluded that Fe(III)EDTA may be a useful compound for food fortification of cereals because the Fe is well absorbed and utilized for haemoglobin synthesis. The substances in cereals which inhibit absorption of simple Fe salts do not appear to inhibit absorption of Fe from Fe(III)EDTA.