Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine.

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine.

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a Phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood, and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.

Decreased glutathione in patients with anorexia nervosa. Risk factor for toxic liver injury?

OBJECTIVE: To investigate glutathione and amino acids related to glutathione metabolism in patients with anorexia nervosa in order to test the hypothesis that these patients exhibit a deficiency of glutathione and therefore might be at an increased risk of developing toxic liver injury. DESIGN: Controlled observatory study and case report. SETTING: University Hospital. SUBJECTS: Subjects included 11 female patients with anorexia nervosa and 12 healthy female controls. INTERVENTIONS: Determination of fasting free and total glutathione, homocysteine, vitamins B(6) and B(12) and folic acid in plasma. RESULTS: A 14-y-old patient with a body mass index of 12.6 kg/m(2) presented with markedly elevated transaminases (ALAT >50 x upper limit of normal), and paracetamol was detected in her blood. Patients with anorexia nervosa exhibited lower circulating concentrations of free cysteine (8.9+/-1.5 vs 12.0+/-1.4 micromol/l) and free and total glutathione (5.0+/-1.3 vs 7.1+/-1.2 and 11.2+/-3.8 vs 16.2+/-5.0 micromol/l, respectively). The plasma concentrations of homocysteine (17.5+/-4.9 vs 12.0+/-3.8 micromol/l) and also of glycine (194+/-37 vs 143+/-41 micromol/l) and glutamine (422+/-51 vs 353+/-51 micromol/l) were significantly higher in patients with anorexia nervosa who were not deficient in folic acid, vitamin B(6) and B(12). CONCLUSIONS: Lower plasma concentrations of glutathione suggest lower intracellular concentrations of the tripeptide. Higher homocysteine, glycine and glutamine concentrations point to a decreased utilization of these amino acids for glutathione synthesis and an impairment of trans-sulfuration. Consequently, the capacity of patients with anorexia nervosa to detoxify electrophilic metabolites and reactive oxygen species via glutathione may be impaired.

Effect of methylene blue on the disposition of ethanol.

The effect of methylene blue on the disposition of ethanol was studied in rats and humans. Methylene blue increased the metabolism of [(14)C]ethanol to (14)CO(2) in isolated hepatocytes and in intact rats by 75% and 30%, respectively. In healthy volunteers, methylene blue did not affect the pharmacokinetics of ethanol and did not alleviate the ethanol-induced NAD redox changes as reflected by the increase in the [lactate]/[pyruvate] ratio.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.

OBJECTIVE: To determine the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is used to prevent ifosfamide-induced encephalopathy in oncology. METHODS: The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application. RESULTS: The time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue. CONCLUSIONS: Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.

Pharmacokinetics of 2-chloro-2'-deoxyadenosine administered subcutaneously or by continuous intravenous infusion.

PURPOSE: Cladribine (2-chlorodeoxyadenosine, 2-CDA) is effective in the treatment of various lymphoproliferative disorders. In the standard protocol the compound is administered by continuous intravenous (i.v.) infusion. In order to allow outpatient therapy alternative modes of administration such as subcutaneous (s.c.) injection would be desirable. The aim of the present study was to compare the pharmacokinetics of 2-CDA after i.v. and s.c. administration. PATIENTS AND METHODS: Nine patients received 0.1 mg/kg 2-CDA per 24 h on one occasion by continuous i.v. infusion and on another occasion as a bolus subcutaneously. The concentrations of 2-CDA in the plasma and urine were determined by HPLC. RESULTS: During i.v. infusion the concentration of 2-CDA in the plasma reached a plateau after 4-8 h, whereas with s.c. administration almost ten times higher peak concentrations were reached within 20 to 60 min. A two-compartment model was fitted to the data points whereby the goodness-of-fit statistics showed R2 values of > 0.98. The calculated rate of elimination, k(elim), averaged 0.336 h(-1) with s.c. and 0.397 h(-1) with i.v. administration. The estimated volumes of distribution were 1.67 and 1.58 l/kg. The areas under the concentration time curves (608 +/- 65 pmol x h/ml after s.c. administration vs 571 +/- 50 pmol x h/ml during i.v. infusion) and the urinary excretion of 2-CDA in 24 h (4.75 +/- 0.95 vs 3.55 +/- 0.53 micromol/24 h) were similar in both groups, indicating identical bioavailability. CONCLUSIONS: Although the pharmacokinetic profile of 2-CDA administered s.c. differs substantially from the profile of a continuous i.v. infusion the areas under the plasma concentration time curves, the urinary excretion of unchanged drug and the estimated pharmacokinetic variables were similar with both modes of administration, indicating that the different time-courses of the plasma concentration did not influence the fraction metabolized or eliminated.

Effect of oral and intravenous S,N-diacetylcysteine monoethyl ester on circulating and hepatic sulfhydryls in the Rat.

The role of GSH in the detoxification of reactive metabolites of oxygen and xenobiotics, in gene expression, and as a source of cysteine is well established. Because decreased circulating and intracellular concentrations of GSH might be of pathogenetic relevance in several clinical conditions, there is a growing interest in pharmacological interventions to correct a deranged sulfhydryl status. In this study, the disposition and the effect of S,N-diacetylcysteine monoethyl ester (DACE) on sulfhydryls were investigated after i.v. and intraduodenal (i.d.) administrations to rats. DACE was rapidly hydrolyzed and deacetylated to N-acetylcysteine and cysteine in plasma. High concentrations of cysteine were attained in the circulation and in the liver after i.v. and i.d. administrations of 5 mmol/kg DACE, and physiological levels of GSH in the liver and in plasma increased by 30 and 300%, respectively, with i.v. and i.d. administrations. Incubation of peripheral blood mononuclear cells with 1 mM DACE resulted in higher intracellular concentrations of cysteine and GSH after 24 h than incubations with equimolar concentrations of cysteine, N-acetylcysteine, or oxothiazolidine carboxylic acid, respectively. It is concluded that DACE provides an efficient delivery system for cysteine that markedly increases intra- and extracellular cysteine and GSH after i.v. and i.d. administrations. Because its uptake into cells is probably not dependent on an active transport process, DACE results in higher intracellular concentrations of cysteine than those resulting from other prodrugs of cysteine and cysteine itself. The compound may thus have advantages over other compounds for the correction of a deranged sulfhydryl status.

Reperfusion injury of the liver: role of mitochondria and protection by glutathione ester.

BACKGROUND: Reperfusion injury of the liver is characterized by intravascular oxidative stress and GSH consumption. Whether mitochondria contribute to hepatocellular damage has never been elucidated. Therefore, we assessed mitochondrial function and redox state during reperfusion and the effect of glutathione monoethyl ester (GSHE) administration, which may replenish the GSH pool. MATERIALS AND METHODS: Rats were subjected to partial hepatic ischemia (90 min) followed by reperfusion. Mitochondrial function was assessed in vivo and in vitro by the KICA breath test and the ATP synthase activity. Just prior to the start of reperfusion, rats received 5 mmol/kg of GSHE or saline iv. ALT, total and oxidized (GSSG) glutathione, GSHE, and CYS were measured in plasma and liver. GSH, GSSG, malondialdehyde (MDA), and carbonyl proteins were measured in mitochondria. The extent of necrosis was also estimated. Sham-operated rats served as controls. RESULTS: Reperfusion markedly increased ALT (>1500 U/L) and doubled the liver content of MDA and carbonyl proteins. Mitochondrial GSH decreased approximately 30%, without increase of GSSG. The in vivo KICA breath test was not significantly impaired by reperfusion. In contrast, both KICA decarboxylation and ATP synthase activity were both reduced by approximately 50% in mitochondria isolated from reperfused livers. GSHE administration significantly decreased ALT ( approximately 40%), protected ATP synthase activity, and reduced the extent of necrosis. Compared to controls, plasma GSHE and plasma GSH at 1 h were lower in rats subjected to ischemia. GSHE was higher in reperfused lobes than in continuously perfused ones and the concentration of GSH was significantly higher in ischemic liver than in untreated animals, indicating that the uptake of GSHE is increased in postischemic liver. GSHE prevented the reperfusion-associated increase of oxidized products in liver and mitochondria. CONCLUSIONS: Reperfusion of ischemic liver is associated with oxidative modifications and functional impairment of mitochondria. GSHE protects against reperfusion injury, possibly by providing intra- and extracellular GSH.

Assessment of liver function prior to hepatic resection.

Improved surgical technique, improved anesthesia, better postoperative care and better selection of patients have all contributed to the marked decrease in mortality of partial hepatectomy over the past few years. Preoperative assessment of portal pressure and of hepatic function with indocyanine green and an oral glucose load can help identify patients who will not tolerate a major hepatic resection. The predictive value of the indocyanine green retention can be improved if the volume fraction of the liver remaining after resection is determined preoperatively by computer tomography. Factors other than hepatic function, such as age, concomitant diseases and characteristics of the operation itself, will of course also play an important role in determining the outcome of partial hepatectomy.

Increased urinary losses of carnitine during ifosfamide chemotherapy.

Chloroacetaldehyde and thiodiglycolic acid, two metabolites of ifosfamide, interfere with mitochondrial function and may sequester carnitine. Urinary excretion of carnitine was measured in five patients before and during a continuous infusion of ifosfamide over 5 days at a dose of 2.8-3.2 g/m2 per day. The excretion of free and total carnitine increased from 85+/-53 to 2697+/-1393 micromol/day on the 1st day of chemotherapy and then gradually decreased. The average loss of carnitine during a chemotherapy cycle amounted to 8.5 mmol. The formation and excretion of esters of carnitine and metabolites of ifosfamide and/or a decreased renal tubular reabsorption could account for this marked loss, which might lead to symptomatic carnitine deficiency after several chemotherapy cycles.

Inhibition and stimulation of long-chain fatty acid oxidation by chloroacetaldehyde and methylene blue in rats.

The effects of chloroacetaldehyde (CAA) and methylene blue, both alone and together, on mitochondrial metabolism, hepatic glutathione content, and bile flow were investigated in rats. Oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [1,4-14C]succinic acid allowed for the differentiation between carnitine-dependent long-chain fatty acid metabolism, medium chain fatty acid oxidation, and citric acid cycle activity, respectively. CAA, a metabolite of the anticancer drug ifosfamide, which may be responsible for ifosfamide-induced encephalopathy, inhibited palmitic acid metabolism but not octanoic or succinic acid oxidation, depleted hepatic glutathione, and stimulated bile flow. Methylene blue, which is clinically used to either prevent or reverse ifosfamide-associated encephalopathy, markedly stimulated palmitic acid oxidation either in the presence or absence of CAA, but did not affect the oxidation of octanoic and succinic acid or hepatic glutathione. Taken together, this study demonstrates that CAA inhibits palmitic acid metabolism. Methylene blue stimulates long-chain fatty acid oxidation, most likely by facilitating the translocation of fatty acids into mitochondria, and compensates for the CAA effect in vivo.

Time course of total cysteine, glutathione and homocysteine in plasma of patients with chronic hepatitis C treated with interferon-alpha with and without supplementation with N-acetylcysteine.

BACKGROUND/AIMS: Glutathione depletion might be one reason for the low rate of response of patients with chronic hepatitis C to treatment with interferon. The aim of the present study was to document the thiol status of patients with chronic hepatitis C and the effects of N-acetylcysteine, a precursor for glutathione synthesis, on the concentrations of total cysteine, glutathione and homocysteine during treatment of chronic hepatitis C with interferon. METHODS: Total cysteine, glutathione and homocysteine in plasma were measured by high performance liquid chromatography, following reduction of disulfides and derivatization of thiols with monobromobimane in a group of 36 patients with chronic hepatitis C, who participated in a multicenter, double-blind, randomized, placebo-controlled clinical trial studying the effect of supplementation with N-acetylcysteine (600 mg three times daily) on the response to treatment with interferon-a (3 MU three times per week) for 6 months. RESULTS: The concentrations of total cysteine (367.0+/-43.9 vs 360.4+/-33.5 nmol/ml, mean+/-95% confidence interval), glutathione (12.5+/-1.6 vs 14.1+/-1.3 nmol/ml) and homocysteine (21.2+/-4.5 vs 19.6+/-5.2 nmol/ml) were similar in patients with chronic hepatitis C and healthy control subjects Supplementation with N-acetylcysteine resulted in measurable concentrations of N-acetylcysteine in plasma, but did not significantly increase the concentrations of cysteine, glutathione or homocysteine. There was no difference between the two treatment groups with regard to transaminases and clearance of HCV RNA. CONCLUSIONS: Circulating concentrations of total cysteine, glutathione and homocysteine are normal in patients with chronic hepatitis C. Supplementation with N-acetylcysteine did not increase the circulating concentrations of total cysteine, glutathione and homocysteine.

[Treatment of alcoholic liver diseases and psychiatric and psychosocial problems]. / Behandlung der alkoholischen Lebererkrankungen und ihrer psychiatrischen und psychosozialen Probleme.

Only about 15% of the subjects abusing ethanol will eventually develop cirrhosis of the liver, suggesting that other factors in addition to the consumption of large quantities of ethanol play a role in the pathogenesis of alcoholic cirrhosis. Important contributors may be infection with hepatitis viruses, in particular HCV, protein-calorie malnutrition and immunologic factors. Abstinence improves the prognosis of patients with alcoholic cirrhosis, provided that the liver disease is not too far advanced. No pharmacotherapeutic intervention has shown a convincing improvement of the prognosis of alcoholic liver disease, so that the therapeutic efforts should be mainly directed towards abstinence. The patient with alcoholic liver disease needs support and guidance by the treating physicians. Supportive treatment with Disulfiram, Acamprosate or Naltrexon can help with achieving durable abstinence.

Thiodiglycolic acid is excreted by humans receiving ifosfamide and inhibits mitochondrial function in rats.

Thiodiglycolic acid has been identified as a major metabolite of the anticancer drug ifosfamide in humans. Patients treated with 12-16 g ifosfamide/m2.day excreted thiodiglycolic acid ranging from 0.10 +/- 0.02 mmol on the first day of therapy, to a maximum of 3.27 +/- 0.15 mmol on the fourth day of ifosfamide infusion. This amounted to 5.4 +/- 0.2% of the administered dose of ifosfamide appearing as thiodiglycolic acid in urine during a 5 days' continuous ifosfamide infusion. Thiodiglycolic acid (50mg/kg) administered to rats inhibited the carnitine-dependent oxidation of [1-14C]palmitic acid by 55%, but affected neither the oxidation of [1-14C]octanoic acid, which is carnitine-independent, nor the oxidation of [1, 4-14C]succinic acid, a marker of Kreb's cycle activity. Additionally, thiodiglycolic acid (30 microM) inhibited oxidation of palmitic acid but not palmitoyl-L-carnitine in isolated rat liver mitochondria, indicating that it either sequesters carnitine or inhibits carnitine palmitoyltransferase I. This study elucidates a specific mitochondrial dysfunction induced by thiodiglycolic acid which may contribute to the adverse effects associated with ifosfamide chemotherapy.

Stimulation of respiration by methylene blue in rat liver mitochondria.

The effect of methylene blue on isolated rat liver mitochondria in the presence and absence of chloroacetaldehyde was investigated. Fatty acid oxidation was inhibited by chloroacetaldehyde and subsequently stimulated by methylene blue. Assessment of tightly coupled mitochondria revealed decreasing respiratory control ratios induced by increasing concentrations of methylene blue and methylene blue provoked mitochondrial swelling. In uncoupled mitochondria, methylene blue promoted a concentration-dependent stimulation of respiration. These findings provide evidence that methylene blue, the redox dye currently used as an antidote for encephalopathy associated with alkylating chemotherapy, uncouples oxidative phosphorylation and acts as an electron transfer mediator to stimulate mitochondrial respiration.

Quantitative liver function in patients with rheumatoid arthritis treated with low-dose methotrexate: a longitudinal study.

The objectives were to determine quantitative liver function prospectively in patients with rheumatoid arthritis (RA) treated with low-dose methotrexate (MTX), to search for risk factors for a loss of quantitative liver function and to assess the relationship between quantitative liver function and histological staging. A total of 117 patients with RA (ACR criteria, 85 women, mean age 59 yr) had measurements of galactose elimination capacity (GEC), aminopyrine breath test (ABT) and liver enzymes [aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (AP), 7-glutamyl transferase (GGT), bile acids, bilirubin, albumin] before treatment with weekly i.m. MTX injections and every year thereafter. In 16 patients, liver biopsies were performed. Before the introduction of MTX, mean GEC was 6.6 mg/min/kg [5th to 95th percentile (5-95 PC) 5.1-8.5; reference range 6.0-9.1] and mean ABT was 0.80% kg/mmol (5-95 PC 0.42-1.30: reference range 0.6-1.0). During treatment with MTX [mean weekly dose 11.8 mg (5-95 PC 5.4-20.2), mean observation period 3.8 yr (5-95 PC 0.4-6.9)], significant declines of GEC (-0.12 mg/min/kg per year. t = 3.30, P < 0.002) and ABT (-0.06% kg/mmol per year, t = 4.81, P < 0.001) were observed. Negative correlations were found between the annual change in GEC and GEC at baseline (Rs = -0.40, P < 0.0001), and the annual change in ABT and ABT at baseline (Rs = -0.43, P < 0.0001). No correlations were found between the annual change in GEC or ABT and weekly MTX dose, age or percentage of increased liver enzymes, and no effect of a history of alcohol consumption > 30 g/week became evident. Two patients with Roenigk grade III had impaired quantitative liver function, while 14 patients with Roenigk grades I and II exhibited a high variability of GEC and ABT from normal to abnormal values. The continuous declines in GEC and ABT observed deserve attention in patients with prolonged treatment. Patients with a low GEC or ABT at baseline seem not to be at increased risk for a further loss of quantitative liver function. An impaired GEC or ABT does not necessarily concur with hepatic fibrosis on histological examination.

Compartmentation of glutathione: implications for the study of toxicity and disease.

The fact that glutathione (GSH) plays many roles in biological protective mechanisms and critical physiological functions has been recognized for decades. Conjugates, disulfides, and other glutathione-derived products also have been studied as biomarkers of the chemical natures or specific identities of key metabolites of toxic agents and such studies have been crucial in the delineation of the nature of the interactions of proximal toxicants with target biomolecules. Despite the extensive evidence implicating the depletion and/or oxidation of glutathione in a wide variety of human and experimental toxicities, critical examination of such studies frequently reveals that injury is not simply related to glutathione status. GSH is compartmentalized at several levels and this compartmentation appears to exert considerable influence on the relationships between glutathione depletion or oxidation and the onset of injury. Although compartmentation is usually viewed from the perspective of different intracellular pools, the significance of extracellular glutathione in functionally important pools is gaining recognition. As the factors affecting the interactions of intracellular pools with extracellular pools are delineated, studies in humans can be designed and interpreted with greater precision and utility.

Decreased release of glutathione into the systemic circulation of patients with HIV infection.

Low glutathione (GSH) in patients with HIV infection could contribute to their immune deficiency since GSH plays an important role in the function of lymphocytes and sulphydryls decrease the expression of HIV in vitro. In order to gain more insight into the mechanisms responsible for the deranged sulphydryl homeostasis in HIV infection, the release of GSH into the circulation, an estimate of the systemic production of GSH, was determined using a pharmacokinetic approach. The basal plasma concentrations of free GSH (3.3 +/- 1.3 vs. 5.3 +/- 1.9 mumol L(-1)) and cysteine (7.7 +/- 2.6 vs. 13.4 +/- 4.9 mumol L(-1)) were significantly lower in eight HIV-infected patients than in eight controls. Upon infusion of GSH at a constant rate of 1 mumol min-1 kg-1, GSH in plasma reached a new plateau. The increment in plasma GSH was significantly larger in the HIV-infected patients than in the controls. The input of GSH into the circulation (12.9 +/- 5.7 vs. 30.1 +/- 11.7 mumol min-1; P < 0.01) and the clearance of GSH (25 +/- 7 vs. 35 +/- 7 mL min-1 kg-1) were significantly lower in patients with HIV-infection. During infusion of GSH the concentration of cysteine in peripheral blood mononuclear cells of the HIV-infected patients increased significantly. Nevertheless, intracellular GSH did not increase. Thus, the consumption of GSH is not increased in HIV infection. Rather, the present data suggest that GSH in patients with HIV infection is low because of a decreased systemic synthesis of GSH.