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Iron-loading diseases remain an important problem because of the toxicity of iron-catalyzed redox reactions. Iron loading occurs in the mitochondria of Friedreich's ataxia (FA) patients and may play a role in its pathogenesis. This suggests that iron chelation therapy could be useful. We developed previously the lipophilic iron chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) ligands and identified 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH) as the most promising analog. Hence, this study assessed the efficacy of PCTH and other PCIH analogs compared with various chelators, including deferiprone and desferrioxamine (DFO). Age- and sex-matched control and FA fibroblasts were preincubated with iron chelators and subsequently challenged with 50 µM H2O2for up to 24 h. The current study demonstrates an interesting structure-activity relationship among the closely related PCIH series of ligands, with only PCTH being highly effective at preventing H2O2-induced cytotoxicity. PCTH increased FA fibroblast cell viability by up to 70%, whereas DFO rescued viability by 1 to 5% only. Hence, PCTH, which was well tolerated by cells was far more effective than DFO at preventing oxidative stress. It is noteworthy that kinetic studies demonstrated PCTH to rapidly penetrate cells to induce 59Fe efflux, whereas DFO, PCIH, 2-pyridylcarboxaldehyde benzoyl hydrazone, and 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone were far slower, indicating it is the rate of chelator permeation that is crucial for protection against H2O2. In addition, PCTH was found to be as effective as or more effective than conventional radical scavengers or the antioxidant idebenone (which has undergone clinical trials) at protecting cells against H2O2-mediated cytotoxicity. These findings further indicate the potential of PCTH for treatment of iron overload.

The reversal of the free layer of a spin-valve with crossed anisotropy was studied by transmission Lorentz electron microscopy. In situ magnetizing experiments were carried out using the Fresnel imaging mode for various applied field orientations. Reversal could be by simple magnetization rotation or by rotation combined with complex domain processes. Asymmetric magnetization reversals whereby the modes on the outward and return paths of a magnetization cycle were seen on occasion. Insight into why the reversal mode varied in the way it did was obtained using a modified Stoner–Wohlfarth model. The model provided a good description of the various modes and there was reasonable agreement between predicted and observed fields at which key stages of the reversals took place. Even though a single variable parameter model of the kind used cannot describe a multi-domain state, its use in inferring the nature of domain configurations that arise is discussed as are its other strengths and weaknesses.

Amino acid and peptide conjugates of protoporphyrin have been prepared by reacting protoporphyringen with cysteine, glutathione and peptides containing a free thiol group under acidic conditions. The conjugates were formed by the addition of the thioamino acids or peptides to the vinyl groups of protoporphyrin during the autoxidation of protoporphyinogen to protoporphyrin and is free‐radical‐mediated. The conjugates were separated by high‐performance liquid chromatography (HPLC) and characterized by HPLC/electrospray ionization mass spectrometry (HPLC/ESI‐MS) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). All the conjugates formed were diconjugates consisting of diastereoisomers. Copyright © 2002 John Wiley & Sons, Ltd.

Nineteen consecutive patients with metastatic or recurrent nasopharyngeal cancer (NPC) receiving combination chemotherapy were monitored for EBV DNA in their serum. EBV DNA (EBER-1) concentration in serum was measured before, during, and after chemotherapy. Thirteen patients had additional multiple prechemotherapy readings. There was a significant lead time from first detection of serum EBER-1 to clinical recurrence in 62 of patients by a mean of 17.4 weeks (range: 8-74.5 weeks; mean 28.2 weeks if confined to the 8 patients with significant lead time). The median EBER-1 concentration was significantly higher in those with distant metastasis as compared to those with loco-regional recurrence only (17,468 vs. 684 pgmL serum; p 0.046 , Mann-Whitney Utest). Among the 13 patients who responded to chemotherapy, 4 exhibited clinical complete remission (CR) who were only found in the group with EBER-1 DNA drop to background level, while the magnitude of EBER-1 drop did not discriminate partial remission (PR) and stable disease (SD) patients clearly. Subsequent profile of EBER-1 DNA showed concordance with clinical course of either continuous remission or later progression. EBER-1 DNA in serum can become a useful adjunctive surrogate marker to monitor chemotherapeutic response in NPC patients with distant metastasis or advanced local recurrence.

The in vivo and in vitro metabolism of temoporfin (m-THPC), one of the most potent photosensitizers for the treatment of cancer by photodynamic therapy, has been studied in detail by HPLC with fluorescence and spectrophotometric detection and on-line HPLC-electrospray mass spectrometry. The results showed that temoporfin was not metabolized in vivo and was excreted unchanged via the biliary system into the faeces. No temoporfin or metabolites were detected in the urine. In vitro incubation of temoporfin with human and rat liver microsomal preparations in the presence of NADPH resulted in no metabolite production, even after enzyme induction with cytochrome P-450 isoenzyme inducers such as phenobarbitone, dexamethasone and 3-methylcholanthrene. No conjugation of temoporfin by phase II cytosolic enzymes was observed. It is concluded that the possible ‘metabolites’ previously observed were artifacts generated by photochemical oxidation of temoporfin to hydroxylated derivatives during the sample administration, collection, preparation and extraction procedures or were impurities already present in the original drug before administration for metabolic studies. These have been confirmed experimentally. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: ES-MS, electrospray mass spectrometry; PDT, photodynamic therapy; m-THPC, temoporfin, 5, 10, 15, 20-tetra (m-hydroxyphenyl) chlorin.

The in vivoand in vitrometabolism of temoporfin (m‐THPC), one of the most potent photosensitizers for the treatment of cancer by photodynamic therapy, has been studied in detail by HPLC with fluorescence and spectrophotometric detection and on‐line HPLC‐electrospray mass spectrometry. The results showed that temoporfin was not metabolized in vivoand was excreted unchanged via the biliary system into the faeces. No temoporfin or metabolites were detected in the urine. In vitroincubation of temoporfin with human and rat liver microsomal preparations in the presence of NADPH resulted in no metabolite production, even after enzyme induction with cytochrome P‐450 isoenzyme inducers such as phenobarbitone, dexamethasone and 3‐methylcholanthrene. No conjugation of temoporfin by phase II cytosolic enzymes was observed. It is concluded that the possible ‘metabolites’ previously observed were artifacts generated by photochemical oxidation of temoporfin to hydroxylated derivatives during the sample administration, collection, preparation and extraction procedures or were impurities already present in the original drug before administration for metabolic studies. These have been confirmed experimentally. Copyright © 1999 John Wiley & Sons, Ltd.

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.

A fast and simple method was developed for the separation of unconjugated bilirubin and its mono- and di-glucuronide conjugates from bile by high-performance liquid chromatography (HPLC). Unconjugated bilirubin was separated on a reversed-phase column using acetonitrile-water (70:30 v/v) as the mobile phase, while the conjugates were separated on a μ-Bondapak-carbohydrate column employing acetonitrile-water (90:10 v/v) as the eluent. The application of this method was demonstrated by the analysis of the bile pigments in rat bile.

A fast, simple and specific method for the determination of ioglycamic acid in bile and in urine is described. Reversed-phase ion-pair chromatography with methanol-water (67.5: 32.5) in the presence of the counter ion tetrabutyl-ammonium phosphate as the mobile phase separates ioglycamic acid in 5 min at a flow rate of 1 ml/min. Under the same conditions urinary creatinine can be simultaneously determined and may be used as an endogenous internal standard. Bile and urine from a healthy subject were screened for possible metabolites but these are not detected.

A reversed-phase high-performance liquid chromatographic method for the determination of quercetin in human plasma following intravenous infusion is described. Quercetin in plasma was extracted with methanol–dimethyl sulphoxide (4:1 v/v) and separated on a C18 Hypersil-BDS column with 44% (v/v) methanol in 0.1 m ammonium acetate (pH 5.15) containing 0.27 mm EDTA as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 375 nm, or electrochemically, with a detection limit of 80 ng/mL and 2 ng/mL, respectively. © 1998 John Wiley & Sons, Ltd.

An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy.

A high-performance-liquid-chromatographic method is developed for the simultaneous determination of hydroxymethylbilane synthase and uroporphyrinogen III synthase activity in erythrocytes. Effective separation of uroporphyrin I and III isomers allows the accurate quantification of individual isomers and the total uroporphyrin concentration. Total uroporphyrin production is used to calculate hydroxymethylbilane synthase activity, and the amount of uroporphyrin III formed represents the activity of uroporphyrinogen III synthase. Normal ranges are established for the two enzymes.

The isomeric compositions of the heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens formed by incubation of porphobilinogen with human red-cell haemolysates have been analysed and compared with those derived from incubation with chemically prepared uroporphyrinogen III as substrate. The results indicated that when supplied with an excess (3.7 microM) of exogenous uroporphyrinogen III, uroporphyrinogen decarboxylase utilized the substrate at random and a mixture of isomers was produced; whereas with uroporphyrinogen III generated enzymically from porphobilinogen as substrate a clockwise decarboxylation sequence was observed, resulting in the formation of intermediates mainly with the ring-D, rings-AD and rings-ABD acetate groups decarboxylated. Using [14C]uroporphyrinogen III as substrate at low concentrations (0.01-0.5 microM) also led to preferential decarboxylation of the ring-D acetate group. It was concluded that the order of uroporphyrinogen III decarboxylation is substrate-concentration-dependent, and under normal physiological conditions enzymic decarboxylation is most probably orderly and clockwise, starting at the ring-D acetate group.

A zero insertion force (ZIF) connector was designed with the requirement of maintaining a specified contact normal force and wipe sufficient to achieve reliable electrical performance. In order to evaluate the design during its development and to monitor its quality during full scale production, a nondestructive sensor was developed to measure the force and wipe simultaneously. The sensor was built into a steel coupon, and assembled in a benchtop fixture that facilitates ease of alignment and measurement of the contact springs. The coupon can also be mounted on a printed circuit card to enable “in-situ” measurement of connectors assembled in the electronic “cage-like” package. Prior to sensor availability, the connector vendors as well as IBM had other methods of measuring contact force and wipe. These measures had varying accuracies, and were difficult to correlate. The sensor described in this paper was duplicated in order for the vendors and IBM to have a consistent measurement tool.

A reversed‐phase high‐performance liquid chromatographic method for the determination of quercetin in human plasma following intravenous infusion is described. Quercetin in plasma was extracted with methanol–dimethyl sulphoxide (4:1 v/v) and separated on a C18Hypersil‐BDS column with 44% (v/v) methanol in 0.1 mammonium acetate (pH 5.15) containing 0.27 mmEDTA as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 375 nm, or electrochemically, with a detection limit of 80 ng/mL and 2 ng/mL, respectively. © 1998 John Wiley & Sons, Ltd.

The separation of toremifene and its metabolites 4-hydroxytoremifene, N-desmethyltoremifene, N-desdimethyltoremifene and deaminohydroxytoremifene by reversed-phase high performance liquid chromatography is described. The effects of pH, buffer concentration and type and proportion of organic modifier on the retention and resolution of the compounds have been studied. This allows optimum conditions for a particular biological application to be developed by simple modification of these paramaters. For the separation of toremifene and metabolites in microsomal metabolism and in plasma, the optimum conditions were 65% (v/v) acetonitrile in 0.25M ammonium acetate-acetic acid buffer, pH 5.0-5.2.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.

A zero insertion force (ZIF) connector was designed with the requirement of maintaining a specified contact normal force and wipe sufficient to achieve reliable electrical performance. In order to evaluate the design during its development and to monitor its quality during full scale production, a nondestructive sensor was developed to measure the force and wipe simultaneously. The sensor was built into a steel coupon, and assembled in a benchtop fixture that facilitates ease of alignment and measurement of the contact springs. The coupon can also be mounted on a printed circuit card to enable “in-situ” measurement of connectors assembled in the electronic “cage-like” package. Prior to sensor availability, the connector vendors as well as IBM had other methods of measuring contact force and wipe. These measures had varying accuracies, and were difficult to correlate. The sensor described in this paper was duplicated in order for the vendors and IBM to have a consistent measurement tool.

The use of adhesive joints as structural elements is increasing rapidly. A thorough understanding of failure behavior of bulk adhesive is essential in analyzing the failure mode of adhesive joints.1 This article discusses existing failure criteria for continua under multiaxial stress fields in most general terms. From this general description, an attempt was made to formulate by simple experiments the specific failure criterion of a typical polyamide cured epoxy adhesive, which is to be an input for the failure analysis of a given adhesive joint system utilizing the same adhesive.

High performance liquid chromatography (HPLC) has been investigated for the separation of the following reduced oligosaccharides containing neutral and acetamido sugars;[image omitted]These alditols were studied as standards for the separation of mixtures of reduced oligosaccharides obtained from glycoproteins.A combination of several HPLC systems using normal and reverse phase column packings was required for separation of the isomers as follows. A novel chromatography system using acetylated alditols eluted from silica (Hypersil) with dichloromethane/ hexane/isopropanol as mobile phase separated the disaccharides and the first trisaccharide from the next two. These last two trisac charides could in turn be separated as non-acetylated alditols chromatographed on Hypersil eluted with aqueous acetonitrile containing 0.05% tetraethylenepentamine (TEPA) or on silica chemically bonded with aminopropyl groups (APS-Hypersil). The pentasaccharides were resolved as acetylated alditols chromatographed on reverse phase, octodecyl silica (ODS-Hypersil).Isomeric separation of the tetrasaccharides was not achieved. However LNT-01 could be obtained essentially free of LNNT-01 by isolation of its di-N-acetylated product. The third tetrasaccharide studied was readily separated from the other tetrasaccharides and the pentasaccharide isomers on normal or reverse phase chromatography because of its greater acetamido/neutral sugars ratio. In general the varying ratios of acetamido/neutral sugars and their different glycosidic linkages conferred distinct but predictable chromatographic properties to the alditols on silica and reverse phase chromatography.

A simple, rapid, sensitive and specific high-performance liquid chromatographic method is developed for the determination of verapamil and its active metabolite norverapamil in human serum. The sample (100 μl) is vortex-mixed with methanol (200 μl) and after centrifugation 100 μl of the supernatent is used for the analysis of a C8 reverse-phase column (10 cm × 5 mm) with methanol - 0.1 M ammonium acetate (55:45 v/v) as the mobile phase. The compounds are detected fluorimetrically with a detection limit of 5 μ/l using an excitation wavelength of 278 nm and an emission wavelength of 320 nm. The method is applicable to pharmacokinetic studies and to therapeutic monitoring of the drug and its active metabolite.

The separation of uroporphyrin I and III isomers by reversed-phase high performance liquid chromatography on ODS-Hypersil with ammonium acetate buffer-acetonitrile solvent systems is described. The effects of buffer concentration, pH, organic modifier proportion and different organic modifiers on the resolution are studied. The optimum conditions for the separation were 12-13% acetonitrile in 1M ammonium acetate buffer pH 5.10-5.20. The method also separated uroporphyrin I and III from the II isomers but the resolution of uroporphyrin III and IV isomers was not achieved.

A reversed-phase HPLC system with 22% (v/v) acetonitrile in 1 M ammonium acetate buffer pH 5.16 as mobile phase on an ODS-Hypersil column is developed for the analysis, isolation and characterization of the pentacarboxylic porphyrins derived from uroporphyrinogen III. The results proved conclusively that enzymic decarboxylation of uroporphyrinogen III does not always begin at the ring D acetic acid group and proceeds in a clockwise manner as currently believed.

Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD). cop homologs were cloned from the chromosome of P. syringae pv. tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P. syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance. Two chromosomal cop homolog regions were cloned. Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS. Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P. syringae. However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC. In addition, homolog 2 conferred a high frequency of mutation to full copper resistance. In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed. pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs. Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.

A novel HPLC system has been developed that has allowed the separation of tamoxifen DNA adducts formed in the livers of rats and mice treated with this drug. At least 13 different peaks have been separated from 32P-post-labelled DNA, with two major peaks jointly accounting for >60% of the total adducts formed by tamoxifen in the livers of treated rats and mice. This is a great improvement on the resolution obtained by thin layer chromatography, which separates the adducts into one main product consisting of a group of major adduct spots eluting together, plus several other minor spots. Identification of the nature of some of the peaks has been investigated. Comparisons of the products formed when alpha-acetoxytamoxifen is reacted with DNA in vitro with 32P-post-labelled liver DNA adducts from rats treated with tamoxifen or alpha-hydroxytamoxifen in vivo, appear to confirm that a major route of activation of tamoxifen in vivo is via alpha-hydroxylation. The resolving power of this HPLC system has further extended this result to show that six of the peaks, including the two major peaks, are formed by the reaction of an activated alpha-hydroxytamoxifen with DNA. Activation of 4-hydroxytamoxifen by the peroxidase/H2O2 system in vitro gives a more polar DNA adduct seen only at trace levels in liver DNA from tamoxifen-treated rats and mice.

A kinematically admissible velocity field is derived for a proposed arbitrarily inclined triangular UBET element. The method is applied to combined forging to show the flexibility of application. From the derived velocity fields upper-bound loads on the punch and deformed configurations are determined by optimizing some given parameters related with geometry and velocity field. Experiments on combined forging are carried out with annealed Al-2024 billets at room temperature for several punch shapes. The theoretical predictions both in the forming load and deformed configuration are in good agreement with the experimental results. It is shown that arbitrarily inclined triangular elements proposed in this work can effectively be used for the prediction of the forming load and deformation in combined forging and can be applied to other forming processes with flexibility.

The in vitrometabolism of toremifene has been studied in liver microsomal preparations from rat, mouse and human sources using high‐performance liquid chromatography–electrospray ionisation mass spectrometry (HPLC–ESIMS). The metabolites detected were N‐desmethyltoremifene (m/z392), 4‐hydroxytoremifene (m/z422), 4′‐hydroxytoremifene (m/z422) and toremifene N‐oxide m/z422). In addition, a new polar metabolite with a protonated molecule at m/z422 has been detected in all three species. The compound was identified by tandem MS‐MS as α‐hydroxytoremifene, an analogue of α‐hydroxytamoxifen. The results showed that α‐hydroxylation is a common feature of tamoxifen and toremifene metabolism and that α‐hydroxytamoxifen is unlikely to be the reactive metabolite responsible for the hepatocarcinogenesis in rat, as widely believed. Copyright © 2002 John Wiley & Sons, Ltd.

No Abstract.

A reversed-phase h.p.l.c. system is described for the separation of all five naturally occurring pentacarboxylic porphyrinogen isomers. The compounds are detected electrochemically with high sensitivity. The peaks are positively identified by h.p.l.c. analysis of the pentacarboxylic porphyrinogens from reduction of pentacarboxylic porphyrins prepared by partial decarboxylation of hexa- and hepta-carboxylic porphyrin III of known structures. The resolution of pentacarboxylic porphyrinogens is superior to that of the porphyrins and the method is applicable to the small-scale preparative isolation of pure isomers.

The isomeric composition of type-III heptacarboxylic porphyrinogens derived from decarbosylation of uroporphyrinogen III by erythrocyte uroporphyringogen decarboxylase was analysed by h.p.l.c. with electrochemical detection. All four possible isomers were identified, and there were little differences in the proportion of isomers formed by erythrocytes from normal subjects and from patients with sporadic porphyria cutanea tarda. The results provide conclusive evidence that the normal decarboxylation pathway is random in nature, and the fourth isomer only increases when enzyme abnormality is found.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.

A new porphyrin, meso-hydroxyuroporphyrin I, was isolated from the urine of a patient with congenital erythropoietic porphyria. The structure was characterized as the methyl ester, ethyl ester and acetoxy derivatives by fast-atom-bombardment m.s., by conversion into uroporphyrin I and by chemical synthesis.