Total homocysteine levels in plasma: high-performance liquid chromatographic determination with electrochemical detection and glassy carbon electrode.

An isocratic high-performance liquid chromatography with electrochemical detection (HPLC-ED) method for the determination of total plasma homocysteine [H(e)] has been developed. The electrochemical detection is performed using a glassy-carbon electrode that is not specific for thiol groups. We have tried to solve the problem of specificity focusing our work on chromatographic resolution and have obtained good results without coelution of other thiol compounds or any substances mentioned as common interferences for carbon electrode methods: uric acid, ascorbic acid and salicylates. Thirty samples a day can be assayed for total homocysteine with a lower limit of detection of 2 pmol, and a limit of quantification of 1.0 micromol/l, with a coefficient of variation (C.V.) <20%. For a concentration of total plasma homocysteine of 9.36 micromol/l, the intra- and inter-assay C.V.s were of 3.86% and 5.55% respectively. The analytical recovery achieved in the preparation of the samples ranged from 85.0% to 98.3% and the electrochemical response was linear up to 100 micromol/l.

Homocisteína plasmática: factor de riesgo independiente de afecciones vasculares oclusivas / Plasma homocysteine: independent risk factor for occlusive vascular disease

La determinación de los niveles de homocisteína plasmática total se ha convertido en un estudio de gran utilidad debido a que los valores moderadamente elevados de homocisteína circulantes pueden causar aterosclerosis y obstrucción de las arterias coronarias. Se sabe que son varios los factores que causan el aumento de homocisteína plasmática; éstos incluyen afecciones metabólicas hereditarias, estado nutricional y el tratamiento con ciertos fármacos. Los posibles mecanismos por los cuales los niveles elevados de homocisteína causan afecciones vasculares incluyen efectos sobre las plaquetas, los factores de coagulación y el endotelio. Las lesiones ateroscleróticas y la trombosis relacionadas con la hiperhomocisteinemia podrían ser prevenidas con una dieta rica en vitaminas, aunque esto hasta el presente no ha sido comprobado (AU)

Homocisteína plasmática: factor de riesgo independiente de afecciones vasculares oclusivas / Plasma homocysteine: independent risk factor for occlusive vascular disease

La determinación de los niveles de homocisteína plasmática total se ha convertido en un estudio de gran utilidad debido a que los valores moderadamente elevados de homocisteína circulantes pueden causar aterosclerosis y obstrucción de las arterias coronarias. Se sabe que son varios los factores que causan el aumento de homocisteína plasmática; éstos incluyen afecciones metabólicas hereditarias, estado nutricional y el tratamiento con ciertos fármacos. Los posibles mecanismos por los cuales los niveles elevados de homocisteína causan afecciones vasculares incluyen efectos sobre las plaquetas, los factores de coagulación y el endotelio. Las lesiones ateroscleróticas y la trombosis relacionadas con la hiperhomocisteinemia podrían ser prevenidas con una dieta rica en vitaminas, aunque esto hasta el presente no ha sido comprobado

Antiarthritic synergism of combined oral and parenteral chrysotherapy. II. Increased inhibition of activated leukocyte oxygen burst by combined gold action.

We have observed an antiarthritic effect of combined chrysotherapy in adjuvant arthritis. Since superoxide radicals (O2-) are potent mediators of rheumatoid inflammation, we studied the combined effect of auranofin (AF) and injectable golds on luminol-dependent chemiluminescence (LDCL) and O2- generation by cytochrome-c reduction of activated leukocytes by different receptor-mediated stimuli: phorbol myristic acetate, 10(-6) M; f-Met-Leu-Phe, 10(-6) M; and poly-L-histidine, 10(-5) M. AF, 0.6 and 0.9 micrograms Au/ml, inhibited 34 and 58% of O2- generation, respectively; the addition to AF of 0.3 micrograms Au/ml of gold thiosulfate (GTS) increased this inhibition to 84 and 97% of the oxygen burst. Similar synergistic potentiation inhibition was obtained by LDCL. When the inhibition of O2- generation by the combined action of AF and GTS was compared with AF + gold sodium thiomalate (GTM), only GTS showed an activation on AF's inhibition of the oxygen burst of human leukocytes. The ligand thiosulfate in equimolar concentrations to GTS had a statistically significant (P less than 0.01) inhibitory effect on AF's blockade of O2- generation during the first 5 min of the interaction with the PMNs; thiomalate had no effect. Sequential pretreatment of PMNs with AF and GTS on O2- generation revealed that for synergism of combined gold action to take place, the cell membrane had to be subjected first to the action of oral gold or to the simultaneous combined action of oral and parenteral gold.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiarthritic synergism of combined oral and parenteral chrysotherapy. I. Studies in adjuvant-induced arthritis in rats.

In comparative clinical studies of auranofin (AF, oral gold) and parenteral gold in the treatment of rheumatoid arthritis, no difference in efficacy was detected. Since the pharmacologic profiles of these compounds are different, we studied their combined effect on adjuvant arthritis (AA). The effect of AF alone and combined with gold sodium thiomalate (GTM) or gold sodium thiosulfate (GTS) on the excretion of urinary hydroxyproline (UHP) and urinary calcium (UCa), and the articular index of arthritic rats was followed during five weeks of treatment. The excretion of UHP and UCa was significantly inhibited (P less than 0.005) in rats treated with AF combined with GTM or GTS as compared with animals treated with the individual gold compounds. However, the articular index only decreased significantly (P less than 0.02) in the group of rats treated with AF + GTS. The present studies open the possibility that combined treatment with oral and injectable gold provide a new approach for chrysotherapy with an increased antiarthritic potency.

The effect of gold salts on casein-induced macrophage activation and serum amyloid protein SAA levels.

Previous work from our laboratory has demonstrated a marked inhibitory activity of Auranofin (Au) and Gold Sodium Aurothiomalate (GST) on monocyte-macrophage function and an important role for macrophages in the pathogenesis of casein-induced experimental amyloidosis. In the present study we have looked at the in vivo effect of Au and GST on the inhibition of casein-induced macrophage activation and serum SAA levels. Au and GST markedly inhibited casein-induced macrophage activation while only Au reduced serum SAA levels to a significant degree. The present data raises the possibility that Au may be helpful in the treatment of secondary amyloid disease.

Superoxide radical production by human leukocytes exposed to immune complexes: inhibitory action of gold compounds.

Superoxide radicals produced by phagocytic cells are considered to be important mediators in the rheumatoid inflammation. The effect of the gold compounds auranofin (AF) and gold sodium thiomalate (GST) on superoxide production by human leukocytes was investigated in two models of immunologic injury: immune-complex phagocytosis and frustrated phagocytosis. In both systems, AF (0.5-1.0 micrograms Au/ml) showed a potent inhibitory activity on superoxide generation, quantitated by ferricytochrome c and NBT reduction. GST showed only modest inhibition at higher concentrations (100 microM). The thiol protecting agent dithiothreitol, 1 mM, completely blocks the inhibitory effect of AF. The inhibition of the oxy radical generation by AF may play an important role in the control of rheumatoid inflammation; it is suggested that this action might be mediated through sulfhydryl-AF interaction at the cellular membrane level.

The effect of auranofin and sodium aurothiomalate on peripheral blood monocytes.

The effect of oral and parenteral gold preparations on the functional capacity of human peripheral blood monocytes was studied in various in vitro systems. Both agents were capable of inhibiting monocyte chemotaxis, expression of Fc and C3 receptor sites and to a lesser extent the reduction of nitroblue tetrazolium dye. No difference was observed in the effect of oral or parenteral gold on normal and rheumatoid monocytes. The inhibition of monocyte function by gold salts was associated with an increase in intracellular cyclic AMP levels. Our data suggests that the beneficial effect of gold salts in rheumatoid arthritis may result from its action on the functional activities of the monocyte-macrophage system.

Studies of the effect of auranofin, a new antiarthritic agent, on platelet aggregation.

Auranofin (AF), at a concentration of 10 micrograms/ml, was found to be a potent inhibitor of ADP-, epinephrine-, or collagen-induced platelet aggregation utilizing platelet-rich plasma obtained from human blood. In contrast, aurothioglucose was less effective than AF in inhibiting epinephrine- or collagen- induced platelet aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation and was a function of drug preincubation time. Compared to platelet-rich plasma, washed platelets were superior for detecting the inhibitory action of AF (greater than or equal to 0.1 microgram/ml) on ADP-induced platelet aggregation. This potent inhibitory action of AF on ADP-induced platelet aggregation was antagonized by dithioerythriol, a potent reducing agent. These results suggest that AF can inhibit both platelet release and aggregation mechanisms which may be relevant to its antiarthritic activity. Further studies are required to elucidate the cellular mechanism by which AF inhibits platelet aggregation.

Natural inhibitors and fibrinogen/fibrin degradation products in patients with myocardial infarction.

The activity of the inhibitor of activated Stuart factor (anti-Xa) was evaluated together with the activity of the antiactivator of plasminogen (antiplasminogen) and the amount and nature of the fibrinogen/fibrin degradation products (FDP), as indicators of the existence of the risk for hypercoagulability in patients with acute and recent myocardial infarction (AMI and RMI, respectively). The activity of anti-Xa was diminished in the AMI group with a p less than 0.001. The antiplasminogen activity was increased in both groups with a p less than 0.001; on the other hand, the amount of FDP was increased in the AMI patients and they were of the 'early' type, while in the RMI group the increase was not as marked, but the FDP were of the 'late' type. In both groups the euglobulin lysis time was very prolonged, while the plasminogen did not vary significantly. The tests described appear to be valuable tools for studying the status of the cardiovascular system during cardiac rehabilitation.

Oral chrysotherapy in rheumatoid arthritis: minimum effective dose.

We studied the dose-response to a new oral gold compound in 28 patients with definite rheumatoid arthritis, divided in 4 groups of 7 patients, each treated with different doses of auranofin for 3 months. Clinical and laboratory parameters were recorded weekly, and blood gold levels (BGL) measured by atomic absorption spectroscopy. Six and 9 mg daily doses of auranofin were most effective based on clinical and laboratory results. Correlation studies between BGL and percent decrease of humoral measurements, within the 3 months were statistically were statistically significant. Mean BGL, associated with clinical improvement, reached 0.73 microgram/ml, and was accompanied by a 17.6% decrease from initial value of IgG, 17.1% of alpha 2-globulin, 48.9% of RF titer and 25.9% of ESR.

Effect of auranofin, a new antiarthritic agent, on immune complex-induced release of lysosomal enzymes from human leukocytes.

Auranofin, an oral chrysotherapeutic agent effective in the treatment of rheumatoid arthritis (RA), was found to be a potent, noncytotoxic inhibitor of IgG-RF immune complex-induced lysosomal enzyme release (LER) from human leukocytes. At a concentration of 1 microg Au/ml (5 microM), auranofin produced a marked reduction in beta-glucuronidase (100%), acid phosphatase (88%), and lysozyme (72%) release. In contrast, gold sodium thiosulfate (GST, an injectable gold compound) had no inhibitory activity on LER at equivalent gold concentrations (i.e., 1 microg Au/ml) and only modest activity (less than 36% inhibition) at concentrations as high as 40 microg Au/ml. The 50% inhibitory dose (LD50) of auranofin on LER was calculated to be 3-4 microM (0.6-0.8 microg Au/ml). Blood gold levels in auranofin-treated RA patients were found to be within the range required for in vitro inhibition of LER, and correlated with decreases in IgG, RF titers, and IgG-RF immune-complex formation in vitro. These results suggest that the therapeutic action of auranofin may be caused, at least in part, by inhibition of LER and/or decreases in immune-complex formation.

Effect of auranofin on DNA and protein synthesis in human lymphocytes.

The effect of auranofin-a new oral gold compound for the treatment of rheumatoid arthritis-and gold sodium thiosulphate on DNA and protein synthesis, as well as their effect on membrane transport in stimulated lymphocytes was studied. It was found that only auranofin in the given concentrations inhibits the incorporation of 3H thymidine and 14C amino acids. The studies on membrane transport present evidence that the pharmacological action of auranofin might be mediated through its action at the cellular membrane level.

Auranofin. New oral gold compound for treatment of rheumatoid arthritis.

Eight patients with rheumatoid arthritis were treated with SK & F D-39162 (auranofin), a new oral gold compound which was effective in suppressing adjuvant-induced arthritis in rats. Clinical and humoral parameters were studied during a 3-month period of drug administration followed by a 3-month period under placebo. The drug was absorbed, well tolerated, and its action was manifested by a drop in the mean IgG blood levels in the third week of treatment accompanied by clinical improvement after 5 weeks of oral gold intake. Together with IgG changes, an increase of the albumin ratio was observed, as well as a decrease of alpha2-globulin and rheumatoid factor titres. From a total number of 60 swollen joints found initially in the 8 patients only 17 were swollen at week 12 and 9 at week 15. Although the number of patients treated was too small to allow definite conclusions, a follow-up study under placebo of clinical and laboratory changes in the same patients during another 3-month period showed that IgG serum levels rapidly reverted preceding a flare up of disease activity after withdrawal of the drug. This confirmed a direct role in cause-effect relation played by the new oral gold compound.