High-performance chelation ion chromatography (HPCIC) was employed to retain cationic Cr(III) on an anion-exchange column and hence allow the separation of the two most prevalent forms of chromium, Cr(II) and Cr(VI). A mobile phase of nitric acid was utilized at pH = 1.5; additionally, 2,6-pyridinedicarboxylic acid was used at a concentration of 6 mM. Additives with different structural characteristics were used in an effort to elucidate retention mechanisms. Inductively-coupled plasma mass spectrometry was used for chromium detection. A collision cell was utilized to reduce chloride-based polyatomic ions that may interfere with the detection of Cr(III), and a detection limit study yielded levels in the low part-per-billion range. The newly developed method was applied to the chromatographic analysis of samples of an incubation medium containing Cr(VI) incubated with cell nuclei.

A combined allosteric and competitive model describes the interaction between extracellular Na+ and Rb+ during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on 86Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na+, only a single transport site for extracellular Rb+ exists. After the occupation of the Na+-specific allosteric site, the Rb+ transport pocket opens to allow occupation by an additional Rb+ and the subsequent transport of the two Rb+ ions into the cells. Na+ can also directly compete with Rb+ for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The α3* isoform has an increased intrinsic affinity for Rb+ and a lower affinity for the allosteric Na+ site than α1 or α2*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb+ of inhibitory Na+ bound to the transport site.

Having identified dicyanogold(I) as a common metabolite of gold-based antiarthritis drugs, we are investigating the effects of the compound on the production of lymphokines. Handel, et al. 1 suggested that the transcription factor AP-1, critical to the production of a number of cytokines, might be the target for gold compounds because of a critical cysteine within its DNA binding region. Using Jurkat cells, an established cell line as a model for CD4(+) lymphocytes, we have shown that dicyanogold inhibits the binding of AP-1 to DNA and inhibits the synthesis of IL-2 mRNA and protein. In a macrophage line, THP-1, which synthesizes IL-1beta in response to mitogen, we have shown that dicyanogold inhibits the binding of a second transcription factor, CREB to DNA. Incubation of THP-1 cells with dicyanogold inhibits the production of IL-1beta mRNA. These results suggest that the mechanism of action of gold drugs may be through their interaction with transcription factors necessary for the immune activation seen in Rheumatoid Arthritis.

A competition assay of 86Rb+ uptake in HeLa cells transfected with ouabain-resistant Na(+)-K(+)-ATPase mutants revealed a stimulation of 86Rb+ uptake at low external concentrations (1 mM) of competitor (K+). Of the models that were tested, those that require that two K+ be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat alpha 2 had lower apparent affinities for the first K+ bound than did wild-type rat alpha 2 modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K+ bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K+ bound had a higher flux than those with one K+ bound. Increases in external Na+ decreased the apparent affinity for K+ for all models and decreased the ratio of the apparent influx rate constants for E327L.

Cisplatin is an extremely effective cancer chemotherapeutic agent, but its use is often accompanied by toxicity. Second generation drugs such as carboplatin are becoming more widely used because of reduced toxicity. Since biotransformation products have been implicated in the toxic responses, we have begun to investigate the reactions of cisplatin and carboplatin with potential biological ligands. Reaction products were characterized using HPLC with inductively coupled plasma - mass spectrometry (HPLC-ICP-MS), (1)H and (13)C NMR and fast atom bombardment - mass spectrometry (FAB-MS). Three Pt-creatinine complexes, cis-[Pt(NH(3))(2)Cl(Creat)](+), cis-[Pt(NH(3))(2)(H(2)O)(Creat)](2+) and cis-[Pt(NH(3))(2)(Creat)(2)](2+), were synthesized and the platinum was shown to coordinate to the ring nitrogen, N(3). Human urine samples from patients on cisplatin chemotherapy were shown to contain cisplatin, its hydrolysis product and biotransformation products containing Pt-creatinine, Pt-urea and Pt-uric acid complexes. Urine from carboplatin patients shows fewer biotransformation products. Studies with control and diabetic (protected against cisplatin toxicity) rats showed systematic differences in the biotransformation products formed on administration of cisplatin.

A competition assay ofRbuptake in HeLa cells transfected with ouabain-resistant Na-K-ATPase mutants revealed a stimulation ofRbuptake at low external concentrations (1 mM) of competitor (K). Of the models that were tested, those that require that two K be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat α had lower apparent affinities for the first K bound than did wild-type rat α modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K bound had a higher flux than those with one K bound. Increases in external Na decreased the apparent affinity for K for all models and decreased the ratio of the apparent influx rate constants for E327L.

Gold distribution and binding sites in blood and red blood cells (RBCs) have been determined. RBCs were separated from plasma and lysed. The cytosol was separated from membranes which were then solubilized via detergents. Total gold in each fraction was measured via flow injection analysis (FIA) with inductively coupled plasma mass spectroscopy (ICP-MS) detection. Various high-performance liquid chromatography (HPLC) techniques such as ion-pairing, reversed-phase and size-exclusion chromatography have been applied to RBC samples prepared by incubation with specific compounds and to RBCs from rheumatoid arthritis (RA) patients. Preliminary studies of RA patients' samples indicate very different gold uptake into RBCs depending on the particular patient. Size-exclusion chromatography indicates that gold in the lysate is not bound principally to hemoglobin but rather to a significantly higher molecular weight species (about 330 000 Da). Low molecular weight species in the ultrafiltered RBC lysate include the dicyanogold(I) anion and possibly the bis(glutathione)gold(I) complex. Incubation experiments have been designed to measure dicyanogold(I) and gold drug uptake by RBCs. Experiments with 4,4′-diisothiocyanatostilbene-2,2′-d,l-sulfonic acid (DIDS), an anion channel blocker, indicate that dicyanogold(I) enters the cell by some path other than the anion channel. The inhibition of gold uptake on addition of free cyanide suggests that the loss of cyanide from dicyanogold(I) is important in dicyanogold(I) uptake by RBCs. Given the rapid uptake of dicyanogold(I) and its apparently high tolerance in humans, this material is suggested as a possible therapy in the treatment of AIDS.

We have determined the framework structure of Myochrysine (disodium gold(I)thiomalate) in the solid state and extremely concentrated aqueous solution, previously. It consists of an open chain polymer with linear gold coordination to two thiolates from the thiomalic acid moieties which bridge between pairs of gold atoms providing an Au-S-Au angle of 95 degrees . The question remained: was this structure relevant to the dilute solutions of drugs administered and the still lower concentrations of gold found in the bodies of patients (typically 1 ppm Au in blood and urine or 5 muM in Au). We have provided an answer to that question using extended X-ray absorption spectroscopy (EXAFS) and capillary zone electrophoresis (CZE). EXAFS studies confirm that the polymeric structure with two sulfur atoms per gold atom persists from molar concentrations down to millimolar concentrations. CZE is able to separate and detect Myochrysine at millimolar levels. More importantly, at micromolar levels Myochrysine solutions exhibit identical CZE behavior to that measured at millimolar levels. Thus, aqueous solutions of the drug remain oligomeric at concentrations commensurate with those found in patient blood and urine.The reactivity of Myochrysine with cyanide, a species especially prevalent in smoking patients, was explored using CZE. Cyanide freely replaces thiomalic acid to form [Au(CN)(2)](-) and thiomalic acid via a mixed ligand intermediate. The overall apparent equilibrium constant (K(app)) for the reaction is 6x10(-4)M(-1). Further reaction of [Au(CN)(2)](-) with a large excess of L, where L is cysteine, N-acetylcysteine, or glutathione, shows that these amino acids readily replace cyanide to form [AuL(2)](-). These species are thus potential metabolites and could possibly be active forms of gold in vivo. That all of these species are readily separated and quantified using CZE demonstrates that capillary electrophoresis is an accessible and powerful tool to add to those used for the study of gold-based antiarthritis drugs.

We have shown that dicyanogold(I), [Au(CN)(2)](-) is a common metabolite found in blood and urine samples of patients treated with different gold based drugs. Some patients have high levels of gold within their red blood cells (RBCs). Size exclusion and C18 reversed phase chromatography show that the majority of the gold in RBC lysates is bound to protein, but small molecules such as dicyanogold(I) and gold-glutathione complexes are also present. Dicyanogold incubation with red blood cells in vitro leads to a rapid and complete uptake of gold. This uptake is unaffected by DIDS, an inhibitor of the anion channel, but is blocked by the addition of external cyanide. Dicyanogold is also readily taken up by H9 cells, a continuous CD(4+) cell line. This uptake is significantly inhibited by N-ethylmaleimide, suggesting a requirement for sulfhydryl groups. Dicyanogold inhibits the replication of the AIDS virus, HIV, in a cell culture model.

Our analyses of blood and urine samples from patients treated with gold-based antiarthritis drugs have shown that the dicyanogold(I) anion, [Au(CN)2]', is a common metabolite.Previous work by Graham had suggested that [Au(CN)2]" would form as a result of interaction of any of the gold- based drugs with cyanide and that [Au(CN)2]" would be readily taken up by cells.Our own work with red blood cells indicates that in vitro incubation with [Au(CN)2]" leads to rapid uptake of gold.

Gold compounds are among the few therapeutic agents that can cause remission of aggressive rheumatoid arthritis. Although the mechanism for their toxicity and efficacy is unknown, gold levels in blood have been emphasized recently. In this report, we describe a systematic method to study the gold interaction with red blood cells and its distribution in the cell. Flow injection analysis (FIA) and high performance liquid chromatography HPLC) with gold-specific inductively coupled plasma-mass spectrometry (ICP-MS) detection are used.

Richard Beard, Drexel University Richard Elder, University of Cincinnati Ernst Gleichman, Heinrich-Heine University, Dusseldorf Colin Lock, McMaster University, Ontario, Canada Peter Sadler, Birkbeck College, University of London, UK Frank Shaw, University of Wisconsin-Milwaukee Ewen Smith, Strathclyde University, Glascow, UK Martin Stillman, University of Western Ontario, Canada Katherine Tepperman, University of Cincinnati

A sensitive method is described for measuring cisplatin and some possible metabolites. The method combines reversed-phase ion-pairing liquid chromatography (LC) with inductively coupled plasma mass spectrometry (ICP-MS) for platinum-specific detection. Separation conditions for cisplatin hydrolysis products and the reaction products of cisplatin with methionine, cysteine, and glutathione have been investigated with sodium dodecylsulfate or heptanesulfonate as the ion-pairing agent. The detection limit for cisplatin was found to be 0.1 ng, corresponding to a concentration detection limit of 1 ng/ml when using an injection volume of 100 μl. This study has demonstrated the usefulness of LC—ICP-MS for cisplatin metabolism studies.

Gold based drugs and their metabolites have been characterized using reversed phase, ion pairing chromatography with an inductively coupled plasma mass spectrometer as an element specific detector. For a patient receiving gold sodium thiomalate the principal gold species in the urine is [Au(CN)2]-, which is also seen in a low molecular weight infiltrate of the blood. The same compound is also identified in the urine and blood of a patient taking auranofin and in patients taking solganol. This represents the first identification of a specific gold metabolite in biological fluids taken from patients undergoing gold therapy and the first evidence that different gold drugs have common metabolites.

Many of the growing number of metal-based drugs in use or testing, are metabolically active. To separate and characterize both the drugs and their metabolites we use the element-specific detection capabilities of an inductively coupled plasma mass spectrometer interfaced to a high performance liquid chromatograph. Detection limits are good, typically in the 1-10 ppb range for chromatography and ca. three orders of magnitude better than for atomic absorption spectrometry. Examples of size exclusion, reversed phase and weak anion exchange chromatography are given showing multielement detection from a single sample injection. These samples are from patients undergoing gold drug therapy for rheumatoid arthritis. Problems associated with the use of organic solvents are illustrated and solutions to these problems are demonstrated.

Many of the growing number of metal-based drugs in use or testing, are metabolically active. To separate and characterize both the drugs and their metabolites we use the element-specific detection capabilities of an inductively coupled plasma mass spectrometer interfaced to a high performance liquid chromatograph. Detection limits are good, typically in the 1-10 ppb range for chromatography and ca. three orders of magnitude better than for atomic absorption spectrometry. Examples of size exclusion, reversed phase and weak anion exchange chromatography are given showing multielement detection from a single sample injection. These samples are from patients undergoing gold drug therapy for rheumatoid arthritis. Problems associated with the use of organic solvents are illustrated and solutions to these problems are demonstrated.