Guido Crisponi

Iron chelating agents in clinical practice

Abstract The relevance of iron chelators in medicine has increased in recent years. Iron is essential for life but it is also potentially more toxic than other trace elements. This is due to the lack of effective means to protect human cells against iron overload and to the role of iron in the generation of free radicals. To protect patients from the consequences of iron toxicity, iron chelating agents have been introduced in clinical practice. Unfortunately, the ideal chelator for treating iron overload in humans has not been identified yet. The aim of this review is to report the experience with desferrioxamine therapy in patients affected by β-thalassemia major according to: bioavailability; mechanism of interactions with hepatocellular iron: release of iron chelates and their excretion; impact of iron chelation on survival in thalassemia patients and side effects of prolonged therapy. Problems related to the development of non-toxic oral iron chelators are also discussed, with particular emphasis on the preliminary data on usefulness and safety of deferiprone (L1), recently evaluated in different clinical trials. Iron chelating therapy has been introduced, in recent years, even in the therapy of disorders not characterized by iron overload. Here the following new therapeutic indications are discussed: adult respiratory distress syndrome, myocardial ischemia, cancer and malaria.

Uneven hepatic iron and phosphorus distribution in beta-thalassemia

BACKGROUND/AIMS Determination of hepatic iron concentration is crucial in the evaluation of iron-storage disease. Iron content is normally determined in a part of a needle liver biopsy and the value obtained is considered to be representative of the iron concentration in the whole liver. To evaluate the reliability of this procedure, we studied iron distribution in the liver of two beta-thalassemic patients. Since the transport of intracellular iron is mediated by phosphates, we also studied the hepatic phosphorus distribution. METHODS At autopsy, a liver slice extending from the left to the right lobe was divided into 51 and 49 samples, respectively. Each specimen was subdivided into two parts: one of them was paraffin-embedded and utilized for the histochemical detection of iron; the second part was analyzed for iron and phosphorus content by induced coupled plasma atomic emission spectroscopy. RESULTS The histological picture of both livers was characterized by portal and periportal fibrosis associated with iron storage of different degree, without cirrhosis. The mean iron concentration of the liver was 20,631 +/- 4903 micrograms per g of dry tissue (micrograms/g dt) and 13,901 +/- 1976 micrograms/g dt, respectively. A striking variability in iron content between samples was also found: iron concentration ranged from 11,537 to 32,347 micrograms/g dt in the first case and from 6257 to 16,493 in the second case. We even observed regional differences in iron concentration, with a preferential peripheral accumulation in both cases and a tendency of the left compartment of the liver to accumulate more iron in the first case. Histochemical analyses confirmed the uneven iron distribution even at the acinar level, showing iron mainly being stored in hepatocytes and Kupffer cells of zone 1 of the acinus, with decreasing amounts of iron in zones 2 and 3. The mean hepatic phosphorus concentration was 6662 +/- 1300 micrograms/g dt (range: 4348-9947) and 7502 +/- 986 micrograms/g dt (range: 5844-90,282), respectively. The regional distribution of phosphorus was similar to that observed for iron. A strict correlation between iron and phosphorus content was also observed. CONCLUSIONS Our data show that: 1) iron and phosphorus are unevenly distributed in the beta-thalassemic liver, even in the non-cirrhotic stages; 2) a regional pattern of iron and phosphorus distribution is evident, characterized by higher concentrations at the periphery of the liver; 3) the observed uneven distribution of iron and phosphorus implies that their content determined in a small liver sample cannot be considered as absolutely representative of the mean hepatic iron concentration. Therefore, iron concentrations determined in a part of a needle liver biopsy should be interpreted with caution in monitoring the efficacy of the iron-chelating therapy in beta-thalassemic patients.

Uneven hepatic copper distribution in Wilson's disease

BACKGROUND/AIMS Determination of hepatic copper concentration is important in the diagnosis of Wilsons disease. We studied copper distribution in the cirrhotic liver of a patient who died of Wilsons disease. METHODS A liver slice extending from the left to the right lobe was divided into 38 samples. Each sample was analyzed for copper content by Induced Coupled Plasma Atomic Emission Spectroscopy. RESULTS The mean copper concentration in the liver was 1370 micrograms/g dt. A striking variability, up to 2-3-fold, in copper levels was observed between the samples: the copper concentration ranged from 880 to 2100 micrograms/g dt, with significant differences even between adjacent samples. Lobar differences were also observed, with a tendency of the right lobe to accumulate more copper than the left lobe. Histochemical analyses confirmed the uneven distribution of copper even at the acinar level. Copper was mainly stored in periportal hepatocytes (zone 1) and at the periphery of the regenerating nodules. Moreover, we observed some nodules with the majority of hepatocytes full of copper granules, adjacent to areas of parenchyma negative for copper stains. CONCLUSIONS Our data show that: 1) copper is unevenly distributed in Wilsons disease in the cirrhotic stage; 2) a lobar pattern of copper distribution is evident in this case, characterized by a higher copper concentration in the right lobe; 3) the observed lobar pattern is different from that described in the newborn liver, characterized by a higher copper content in the left compartment of the liver; 4) copper content determined in a small liver sample cannot be considered as absolutely representative of the mean hepatic copper concentration. From a practical point of view, our data show that sampling variability deserves more consideration in the diagnosis and in the monitoring of Wilsons disease. The use of hepatic copper concentration in monitoring the efficacy of the copper-chelating therapy may be unreliable, particularly in the cirrhotic stage, because of the patchy distribution of copper, as demonstrated in this study.

Divalent metal–acetate complexes in concentrated aqueous solutions. An x‐ray diffraction and NMR spectroscopy study

Divalent metal–acetate solutions have been investigated by the x‐ray scattering technique and 13C NMR spectroscopy. The aim of this work was to study the complex formation between the divalent metals and a typical organic complexing ligand in concentrated aqueous solutions. The correlation functions and the analysis of the structure functions agree with the literature information that the species present in solution are Me(H2O)2+6 , Me(H2O)6−z(CH3COO)2+−zz , and Me(H2O)6−2z(CH3COO)2+−zz . Good agreement with experimental data is achieved through a model in which the acetate ions are bonded as a monodentate ligand to the Mg, Co, Mn, and Zn cations and as a bidentate to the Cd cation. The cations also possess a second coordination shell of water molecules. Some indications have been obtained supporting the presence of hydration water around the acetate anions.

Effect of substituents on complex stability aimed at designing new iron(III) and aluminum(III) chelators.

The solution equilibria of iron(III) and aluminum(III) with two classes of hard ligands (catechol, salicylic acid and their nitro-derivatives) have been reliably studied by potentiometric, spectrophotometric and NMR spectroscopy. The effect of the nitro substituent on the binding properties of catechol and salicylic acid has been examined thoroughly. The inductive and resonance properties of the substituent that, as expected, lower the basicity of the phenolic and carboxylic groups, lead to a general decrease in both protonation and complex formation constants. This decrease causes an increase in pM of between 0.2 and 1.1pM units for the nitro-substituted salicylates and of about 4 units for 4-nitrocatechol, with a significantly higher chelating efficacy. The influence of the substituent on catechol and salicylic acid is discussed in detail on the basis of conditional constants at pH 7.4.

Bisphosphonate chelating agents: complexation of Fe(III) and Al(III) by 1-phenyl-1-hydroxymethylene bisphosphonate and its analogues

Abstract Bisphosphonate ligands were found to be very efficient chelating agents for both Al(III) and Fe(III) ions. Potentiometric and spectroscopic data allow evaluation of the coordination equilibria in the solutions containing 1-phenyl-1-hydroxymethylene bisphosphonate and its three analogues with Al(III) and Fe(III) ions. At pH below 4 the bis-complexes are formed, while above pH 4 the major species are equimolar, monomeric for Al(III) or dimeric for Fe(III) complexes. The large steric hindrance and high electric charge are the major factors influencing the complex stoichiometry. The formation of the dimeric Fe(III) with μ-oxo or μ-hydroxo bridges were supported by measurements of the magnetic moments using Evans’ method. The stabilities of the complexes formed are higher than those found for deferiprone, L1, used in clinics as a chelating agent.

Brain copper, iron, magnesium, zinc, calcium, sulfur and phosphorus storage in Wilson's disease.

PROJECT Wilsons disease (WD) is an inherited disorder of copper metabolism characterised by juvenile liver cirrhosis and by neurological symptoms. Copper levels in brain in WD have been reported to be 10 to 15 fold normal values, depending on the different brain regions. Being very few data on copper distribution in central nervous system in WD available, it seemed of interest to study the concentration of copper and of other trace elements (Zn, P, Mg, Ca, Fe and S) in the brain of a patient died for WD. PROCEDURE a 56 year old woman affected by WD was admitted to our hospital with signs of hepatic failure and died few days later. At autopsy, a brain slice extending from the left to the right hemisphere was divided in 28 samples. On each sample Copper, Iron, Magnesium, Phosphorus, Sulphur, Zinc and Calcium were determined by Induced Coupled Plasma Atomic Emission Spectroscopy. RESULTS the mean concentration of copper, ranging from 88 to 158 microg/g of dry tissue in all the brain specimens was higher than literature reference values, while that of the other tested elements was considerably lower. CONCLUSIONS 1) In the brain of WD patient examined the status of trace elements was extensively altered. Further studies are necessary to correlate the concentration of trace elements with pathological lesions and with clinical pictures. 2) The elements considered in our study showed an uneven distribution in different brain areas.

Iron(III) and aluminum(III) complexes with hydroxypyrone ligands aimed to design kojic acid derivatives with new perspectives

With the aim to design new chelators for the clinical treatment of different diseases involving the trivalent metal ions Fe(III) and Al(III), we present the equilibria of kojic acid and its derivative 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one with these two metal ions. Potentiometric and spectrophotometric techniques for iron, and potentiometry and (1)H NMR for aluminum were used, supported by X-ray, electrospray ionization-mass spectrometry (ESI-MS), calorimetry and quantum chemical calculations. In this work, evidence is given on the formation of MeL, MeL(2), and MeL(3) complexes of both metal ions with kojic acid, confirmed by the X-ray structure of the FeL(3) complex, and of variously protonated Me(2)L(2) and MeL(2) complexes of 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one. The extremely good pFe value for this second ligand gives confidence to, and opens perspectives for, the search of new kojic acid derivatives.

Simultaneous decomposition of several spectra into the constituent Gaussian peaks

A computer program is proposed for the decomposition of spectra into their constituent Gaussian peaks. A new feature of this program is the possibility of operating on different spectra from two or more components of variable concentration at the same time, by optimising the wavelengths of the maxima and the halfbandwidths of all the spectra while the height of each peak is optimized in each single spectrum. This distinct treatment is possible due to the fact that the Gaussian functions are linear with respect to the heights and non-linear with respect to the remaining parameters. A linear and non-linear least-squares analysis, respectively, is therefore possible. Besides its intrinsic utility in handling spectral data of different origin this procedure proves more useful than the single spectrum decomposition both in increasing the reliability of the parameters and in improving the convergence properties of the procedure.

Reliability of association constants of 1:1 molecular complexes from spectrophotometric data

Abstract Some basic factors affecting the reliability of K (association constant) and ϵ (molecular extinction coefficient) estimates for 1:1 molecular complexes are studied. To accomplish this, the error matrix is examined and a new variable G=K−1((a+b+K−1)2−4ab)− 1 2 (where a and b are the concentrations of the reagents) is introduced. Whenever G has the same value for all the experimental points, K and ϵ are undetermined. A great dispersion of G values, on the contrary, contributes to more reliable estimates. The variable G has in fact the same role as the independent variable in a linear relationship; therefore the problem of the optimal choice of experimental points is reduced to one of a simple linear regression.