The mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes.

Formation of beta-hematin in acidic acetate solution has been investigated using quantitative infrared spectroscopy, X-ray diffraction, and scanning and transmission electron microscopy. The process occurs via rapid precipitation of amorphous (or possibly nanocrystalline) hematin, followed by slow conversion to crystalline beta-hematin. Definitive evidence that the reaction occurs during incubation in acetate medium, rather than during the drying stage, is provided by X-ray diffraction and infrared spectroscopy of the wet material. The reaction follows a sigmoidal function indicative of a process of nucleation and growth and was modeled using the Avrami equation. Reaction rates and the dimensionality of growth (as indicated by the value of the Avrami constant) are strongly influenced by stirring rate. The reaction follows Arrhenius behavior, and there is a strong dependence of both the rate constant and the Avrami constant on acetate concentration. Acetate may act as a phase transfer catalyst, solubilizing hematin and facilitating its redeposition as beta-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming beta-hematin. The formation of beta-hematin closely parallels many mineralization processes, and this suggests that hemozoin formation may be a unique biomineralization process. Inferences are drawn with respect to the formation of hemozoin in vivo.

Structure-function relationships in chloroquine and related 4-aminoquinoline antimalarials.

Recent publication have provided strong evidence that activity and cellular uptake of 4-aminoquinoline antimalarials depends on vacuolar haemoglobin degradation and that haematin is the drug target. Studies on haematin-quinoline interactions have provided insight into the structural requirements for these interactions and indications are that 4-aminoquinolines may act by inhibiting haemozoin formation. Structural requirements for this activity have also been reported recently and have led to construction of an empirical structure-function relationship for 4-aminoquinolines.

Structure-function relationships in aminoquinolines: effect of amino and chloro groups on quinoline-hematin complex formation, inhibition of beta-hematin formation, and antiplasmodial activity.

Comparison of 19 aminoquinolines supports the hypothesis that chloroquine and related antimalarials act by complexing ferriprotoporphyrin IX (Fe(III)PPIX), inhibiting its conversion to beta-hematin (hemozoin) and hence its detoxification. The study suggests that a basic amino side chain is also essential for antiplasmodial activity. 2- And 4-aminoquinolines are unique in their strong affinity for Fe(III)PPIX, and attachment of side chains to the amino group has relatively little influence on the strength of complex formation. Association with Fe(III)PPIX is necessary, but not sufficient, for inhibiting beta-hematin formation. Presence of a 7-chloro group in the 4-aminoquinoline ring is a requirement for beta-hematin inhibitory activity, and this is also unaffected by side chains attached to the amino group. In turn, beta-hematin inhibitory activity is necessary, but not sufficient, for antiplasmodial activity as the presence of an aminoalkyl group attached to the 4-amino-7-chloroquinoline template is essential for strong activity. We thus propose that the 4-aminoquinoline nucleus of chloroquine and related antimalarials is responsible for complexing Fe(III)PPIX, the 7-chloro group is required for inhibition of beta-hematin formation, and the basic amino side chain is required for drug accumulation in the food vacuole of the parasite.

Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs.

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.

Characterisation of synthetic beta-haematin and effects of the antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine on its formation.

Infrared spectroscopy, elemental analysis and X-ray powder diffraction show that the product of 30 min of reaction of haematin in 4.5 M acetate, pH 4.5 at 60 degrees C is identical to beta-haematin prepared in 4.5 M acetic acid at 70 degrees C overnight (pH 2.6). There is no evidence for formation of haem-acetate complex, which could not be isolated, even from 11.4 M acetate solution. The antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine were found to inhibit formation of beta-haematin, while 5-, 6- and 8-aminoquinoline and quinoline were found to have no effect. Quinidine was shown to form a complex with ferriprotoporphyrin IX in 40% DMSO with log K = 5.02 +/- 0.03. Log K values for halofantrine and desbutylhalofantrine are 5.29 +/- 0.02 and 5.15 +/- 0.02 respectively (solutions containing 30% acetonitrile in addition to DMSO to solubilise these drugs), which are both stronger than chloroquine under the same conditions (log K = 4.56 +/- 0.02).

Muscular herniation of the lower extremities.

Fascial defects are encountered infrequently by physicians who manage disorders of the lower extremities. The correct diagnosis of this condition is important, because the treatment is forthright and will bring the patient relief from symptoms. This article reviews the literature and attempts to aid the clinician in understanding this condition and its treatment. Two illustrative cases are presented.

Thermodynamic factors controlling the interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX.

The interaction of a variety of quinoline antimalarial drugs as well as other quinoline derivatives with strictly monomeric ferriprotoporphyrin IX [Fe(III)PPIX] has been investigated in 40% aqueous DMSO solution. At an apparent pH of 7.5 and 25 degrees C, log K values for bonding are 5.52 +/- 0.03 (chloroquine), 5.39 +/- 0.04 (amodiaquine), 4.10 +/- 0.02 (quinine), 4.04 +/- 0.03 (9-epiquinine), and 3.90 +/- 0.08 (mefloquine). Primaquine, 8-hydroxyquinoline, 5-aminoquinoline, 6-aminoquinoline, 8-aminoquinoline, and quinoline exhibit no evidence of interaction with Fe(III)PPIX. The enthalpy and entropy changes for the interaction of quinolines with Fe(III)PPIX, as determined from the temperature dependence of the log K values, exhibit a compensation phenomenon that is suggestive of hydrophobic interaction. This is supported by the finding that the interactions of chloroquine and quinine with Fe(III)PPIX are weakened by increasing concentrations of acetonitrile. Interactions of chloroquine, quinine, and 9-epiquinine with Fe(III)PPIX are shown to remain strong at pH 5.6, the approximate pH of the food vacuole of the malaria parasite which is believed to be the locus of drug activity. Implications for the design of antimalarial drugs are briefly discussed.

The interaction of the heme-octapeptide, N-acetylmicroperoxidase-8 with antimalarial drugs: solution studies and modeling by molecular mechanics methods.

The equilibrium constants for the coordination of quinine (log K = 2.55 +/- 0.02) and 9-epiquinine (log K = 2.42 +/- 0.05) to form a 1:1 complex with the monomeric ferric hemeoctapeptide from cytochrome c, N-acetylmicroperoxidase-9 have been determined in aqueous solution (pH 6.25, 0.1 M phosphate buffer) at 25 degrees C. The electronic spectra of the complexes suggest that coordination to Fe(III) occurs through the 9-alkoxide group, in agreement with previous NMR work on the interaction of quinine with urohaemin-I [I. Constantinidis and J. D. Satterlee, J. Amer. Chem. Soc. 110, 927 (1988)]. By contrast, two chloroquine molecules bind sequentially to N-acetylmicroperoxidase-8 under the same conditions (log K1 = 3.08 +/- 0.04; log K2 = 1.56 +/- 0.10) in an apparently pi- pi cofacial manner. Molecular mechanics techniques have been applied to a study of the structure of these species with an Fe(III)-porphine nucleus, and show that coordination of the 9-alkoxide group to Fe(III) is possible for both quinine and 9-epiquine, with the quinoline ring system virtually parallel to the porphyrin plane. The energy-minimized structure for the interaction of a single chloroquine molecule with Fe(III)-porphine has the quinoline rings parallel to the periphery of the porphyrin ring.

The chemical mechanism of beta-haematin formation studied by Mössbauer spectroscopy.

Spontaneous formation of beta-haematin (malaria pigment) from haematin in acetate solution follows pseudo-zero-order and not autocatalytic kinetics. Acetate appears to facilitate the reaction by solubilizing the haematin and acting as a phase-transfer catalyst, a role which, in vivo, could be fulfilled by carboxylic acids or amino acids.

The iron environment in heme and heme-antimalarial complexes of pharmacological interest.

Mössbauer spectroscopy has been utilized to probe the electronic environment of iron in a number of Ferriprotoporphyrin IX complexes of relevance to malaria. The markedly different iron environments found for the complexes of hemin with quinine, chloroquine, and the Chinese herbal antimalarial artesunate suggest that these compounds act by protecting the heme from polymerization to insoluble hemozoin, and by facilitating the transport of the protected heme to the food vacuole membrane where it is able to exercise its cytotoxic redox catalytic activity. Mössbauer parameters determined here for purified malaria pigment and synthetic beta-hematin confirm the chemical identical-ness of these species. The Mössbauer spectra of the complexes are discussed in light of the proposed structures of the complexes.

Periodate modification of human serum transferrin Fe(III)-binding sites. Inhibition of carbonate insertion into Fe(III)- and Cu(II)-chelator-transferrin ternary complexes.

Periodate modification of human serum transferrin produces a species that binds Fe(III) weakly at pH 7.4 contrary to previous reports that Fe(III)-binding activity is completely lost. Ternary complexes of periodate-modified transferrin and either Fe(III) with nitrilotriacetate (NTA), oxalate, citrate, or EDTA, or of Cu(II) with oxalate could be formed. Peak wavelength maxima of these spectral bands are identical to those reported for native transferrin in the absence of bicarbonate. No carbonate ternary complexes of periodate-modified transferrin with Fe(III), Al(III), Cu(II), or Zn(II) could be formed. Conditional (Fe(NTA)) binding constants (log K) for C- and N-terminal modified sites are 7.33 and 7.54, respectively. The respective extinction coefficients at 470 nm are decreased 45% compared with the native protein. The electron paramagnetic resonance spectrum of the complex closely resembles that of the Fe(III)-NTA ternary complex formed with native transferrin in the absence of bicarbonate. Anions, including bicarbonate, at high concentrations destabilize formation of this Fe(III)-NTA ternary complex, while Fe(III) chelators readily remove the bound Fe(III). Bicarbonate, sulfate, and pyrophosphate still bind to the modified binding sites in the absence of metal although with slightly lower affinity and with lower molar difference absorptivities. Results are interpreted as an inhibition of a crucial protein conformational change by an intramolecular cross-link, preventing formation of the particularly stable metal-carbonate ternary complex from the less stable metal-chelate ternary complex. The method can be used to produce monosited transferrins.

Release of iron from C-terminal monoferric transferrin to phosphate and pyrophosphate at pH 5.5 proceeds through two pathways.

Iron release fro C-terminal monoferric transferrin at pH 5.5 and 37 degrees C was studied as a function of chloride, phosphate, and pyrophosphate concentration. The rate constant for iron release depends linearly on chloride concentration, confirming that anion binding is mandatory for iron release, not only at pH 7.4 as has been previously reported, but also at pH 5.5. The extent of iron release is relatively small (< 20% for 1.0 M chloride). Concentrations of > 0.2 M phosphate are required for complete iron removal, but millimolar concentrations of pyrophosphate effect complete removal. The observed rate constants for iron release to phosphate and pyrophosphate change from one linear dependence to another less steep linear dependence on the concentration of these ligands, providing quantitative evidence that the two-pathway mechanism that we previously proposed for iron release at pH 7.4 persists at pH 5.5. According to this model, the pathway of iron release is determined by the nature of the anion occupying a kinetically significant anion binding site on the protein. The qualitative similarity of the current data with that recently reported for iron release from the transferrin-transferrin receptor complex provides strong support for the contention that the two-pathway mechanism also persists in this complex at low pH and is hence likely to be operative in vivo.

Quinoline anti-malarial drugs inhibit spontaneous formation of beta-haematin (malaria pigment).

Polymerisation of haematin to beta-haematin (haemozoin or malaria pigment) in acidic acetate solutions was studied using infrared spectroscopy. The reaction was found to occur spontaneously between 6 and 65 degrees C, in 0.1-4.5 M acetate and pH 4.2-5.0. The anti-malarial drugs quinine, chloroquine and amodiaquin were found to block spontaneous beta-haematin formation, while the anti-malarially inactive 9-epiquinine and 8-hydroxyquinoline had no effect on the reaction, as did primaquine, a drug which is active only against exo-erythrocytic stages of infection. It is argued that the intra-erythrocytically active anti-malarial agents act by binding to haematin, blocking beta-haematin formation and leaving toxic haematin in the parasite food vacuoles.

The anion requirement for iron release from transferrin is preserved in the receptor-transferrin complex.

Rates of iron release from both sites of free transferrin at pH 7.4 are critically dependent upon ionic strength, because release appears to require binding of a simple nonchelating anion such as chloride to a kinetically active site of the protein. This site is distinct from the synergistic anion-binding site, occupancy of which is required for binding of iron to occur at all. Complexing of transferrin to its receptor also modulates release of iron, but in a more complex fashion. At extracellular pH, 7.4, receptor retards release, but at the pH of the endosome in which release occurs within the cell, 5.6, receptor accelerates release. The present study was undertaken to determine whether the kinetically active anion requirement is maintained at pH 5.6 and whether the effects of anion binding and receptor binding are independent of each other. A spectrofluorometric method was developed to monitor release of iron from C-terminal monoferric human transferrin and its complex with the transferrin receptor. At pH 5.6, as at pH 7.4, profiles of iron release to pyrophosphate from free and from receptor-complexed monoferric transferrin show curvilinear dependence on pyrophosphate concentration, consistent with a previously described kinetic scheme and suggestive of a similar release mechanism in all cases. Furthermore, at pH 5.6 release rates depend upon anion (chloride) concentration in free and in receptor-complexed transferrin as in free transferrin at pH 7.4, extrapolating nearly to zero as chloride concentration approaches zero. The enhancing effect of receptor on release is displayed at all concentrations of chloride tested,indicating that the release-promoting effects of receptor and chloride are independent of each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.

Identification of bile duct stones in patients undergoing laparoscopic cholecystectomy.

To identify patients with common bile duct stones, all patients considered for laparoscopic cholecystectomy in this unit undergo intravenous cholangiography (IVC) with tomography and, more recently, operative cholangiography. To date 100 consecutive patients with symptomatic gallstones have undergone laparoscopic cholecystectomy with no specific exclusion criteria. Eight patients of 100 were found to have duct stones on IVC with one false-positive. These IVC data were compared with data from 52 patients who also had operative cholangiograms performed. One stone was detected on operative cholangiography that was not identified on IVC. No additional information was gained from operative cholangiography. These data suggest that preoperative IVC is adequate for the detection of duct stones in patients considered for laparoscopic cholecystectomy.

Duodenal obstruction in advanced pancreatic carcinoma: how effective is gastroenterostomy in palliation?

Gastro-enterostomy produces variable and often disappointing results in the management of duodenal obstruction due to advanced pancreatic carcinoma. In a series of 51 patients who had palliative surgery for carcinoma of the pancreas five required a gastro-enterostomy. Three of these subsequently had difficulty with gastric emptying. The problem of gastro-enterostomy failure in palliation of pancreatic carcinoma is discussed.

Iliac artery aneurysm in Marfan's syndrome.

A case of iliac artery aneurysm in a young male patient with Marfan's Syndrome is described. The aneurysm was managed by a simple exclusion technique. The occurrence of such an aneurysm suggests that the inherent mural weakness in the syndrome is more widespread in the arterial tree than is generally appreciated. The association of iliac artery aneurysm and Marfan's Syndrome does not seem to have been previously reported.

The effects of anions on the kinetics of reductive elimination of iron from monoferrictransferrins by thiols.

The kinetics of reductive elimination of iron from the human serum monoferric transferrins by thioglycollate (TG), 3-mercaptopropionate (MP), cysteine (Cys), cysteamine (Cym) and 2-mercaptoethanol (ME) have been studied at 37 degrees C using bathophenanthroline sulphonate (BPS) as the ferrous ion acceptor. Analysis of the entire course of the reaction was possible only with thioglycollate since the other thiols cause eventual protein precipitation; in these cases, initial rates were used. The rate of iron release is linearly dependent on thiol concentration at low concentrations of reductant (less than approx. 0.2 M) and increases more rapidly with higher concentrations (up to 0.5-0.75 M). The thiols fall into two distinct groups, with TG, MP and Cys reacting at approx. the same rate, which is an order of magnitude faster than the reaction with Cym and ME. The carboxylate functionality present in the first group may be responsible for the faster reaction rate, by competitively weakening the interaction between the protein and synergistic anion. The pH-dependence of the rate of reductive elimination appears to depend on ionizable functionalities on both the protein and reducing agent. The addition of NaCl, NaClO4, NaHCO3 and Na2HPO4 increases the rate of iron release from the monoferric transferrins. The last two have particularly large accelerating effects and, in the case of the N-terminal monoferrictransferrin, gave saturation kinetics, suggesting that the observed effect is due to conformational changes in the protein caused by binding of ions. The role of the Fe-synergistic anion complex in the transferrins as a 'trapped intermediate' is considered.

Timing of strangulation in adult hernias.

This retrospective study aimed to determine why the incidence of strangulated hernias in adults remains high. Seventy-nine patients presented with clinical evidence of hernia strangulation which required urgent surgery during the period 1979-87. Forty-six patients (58 per cent) had noted a hernia present for at least 1 month before strangulation: 18 (23 per cent) had not reported it to their family doctor, 19 (24 per cent) were known by family practitioners or non-surgical medical personnel to have a hernia but had not been referred for surgical opinion, and nine (11 per cent) had been previously assessed surgically with a view to elective repair. Of these nine, five were considered unfit, three were on waiting lists for operation and one had refused surgery. Thirty-two patients (40 per cent) presented primarily with strangulation within days of developing a hernia. The duration of hernia before strangulation in one patient was unknown. Although an unavoidable number of patients will continue to present with strangulation within days of developing a hernia, the overall incidence could be significantly reduced by greater public awareness of the risks of hernia strangulation and by a policy of immediate patient referral and prompt elective repair.