[Undesired effects of NSAIDS and coxibs]. / Gastrointestinale, kardiale und renale Nebenwirkungen von NSAR und Coxiben. Was ist wirklich gesichert?

Some 15-30% of patients receiving classical NSAID treatment go on to develop gastrointestinal ulcers. In 0.1-0.2%, hemorrhage and perforations occur in the stomach or bowel. The greater the cox-2 selectivity of an analgesic, the lower is its toxicity. The superiority of cox-2 inhibitors in this respect has been shown in comprehensive studies. Since, however, there have been individual reports of an increase in complications under treatment with coxibs, these agents are no longer recommended for use in patients with coronary heart disease and/or cerebrovascular disease. On the basis of the current data, however, it is also to be expected that EMEA and FDA will soon make similar recommendations with regard to the classical NSAIDs.

Increasing sample throughput in pharmacological studies by using dual-column liquid chromatography with tandem mass spectrometry.

A robust novel technology of parallel chromatography combined with tandem mass spectrometry was successfully applied to a biological matrix extract for analyte detection. The presented study shows how only by using an additional isocratic pump, a second column and a 10-port valve the throughput is twice of that of a conventional single column system with the same sensitivity. Analytes and matrix were separated and eluting peaks of the first column were detected while the second column was equilibrated. The system was tested and used for the determination of several drugs, metabolites and endogenous compounds (i.e., propiverine, talinolol, scopolamine and leukotrienes).

Specific and rapid quantification of 8-iso-prostaglandin F2alpha in urine of healthy humans and patients with Zellweger syndrome by gas chromatography-tandem mass spectrometry.

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is currently discussed as a potential index parameter of oxidative stress in vivo. We describe in this article a fully validated gas chromatographic-tandem mass spectrometric method for the quantitative determination of 8-iso-PGF2alpha in human urine. The method is highly specific and requires a single thin-layer chromatographic step for sample purification. Inter- and intraday imprecision were below 8%. Mean inaccuracy was 5.3% for added levels of 8-iso-PGF2alpha up to 2000 pg/ml of urine. We measured highly elevated excretion of 8-iso-PGF2alpha in the urine of children with peroxisomal beta-oxidation deficiency, i.e. Zellweger syndrome, (63.3+/-16.6 ng/mg creatinine) compared to that of healthy children (0.51+/-0.16 ng/mg creatinine) (mean+/-S.D., both n=5). The method could be useful for diagnosing Zellweger syndrome and for investigating the utility of 8-iso-PGF2alpha as a novel marker for oxidative stress in vivo in man.

Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine.

A high-performance liquid chromatographic method for the determination of free reduced cysteine and N-acetylcysteine in human plasma at the basal state and after oral administration of N-acetylcysteine is described. The method is based on acid-catalysed conversion of plasma thiols to the corresponding S-nitroso derivatives by excess of nitrite and their subsequent cation-pairing RP-HPLC with detection at 333 nm. Recovery rates of cysteine and N-acetylcysteine added to human plasma were 94.6 and 99.6%, respectively. Inter- and intra-day precision were below 6%. In healthy humans (n = 5), free reduced cysteine was determined to be (mean+/-S.E.) 10.0+/-0.96 microM. No N-acetylcysteine was detected in plasma of these subjects above the limit of detection (e.g. 170 nM). The method was successfully applied to a pharmacokinetic study on orally administered N-acetylcysteine to healthy volunteers.

High-dose mitomycin C in isolated hyperthermic liver perfusion for unresectable liver metastases.

In order to reduce systemic side effects and increase intrahepatic mitomycin C (MMC) concentrations, isolated hyperthermic liver perfusion (IHLP) has been performed using MMC. This article describes the pharmacokinetics of MMC in IHLP and presents our clinical experience with its use in six patients suffering from unresectable liver metastases. Primary tumors consisted of colorectal carcinomas in three cases, breast cancer in two, and a choroidal melanoma in one. Dosages of MMC varied between 0.5 and 1.0 mg MMC/kg body weight. MMC was added as a bolus directly into the extracorporeal circuit. Intrahepatic temperature was elevated to 40.0-41.0 degrees C by hyperthermic perfusion. MMC concentrations were measured in peripheral blood (preperfusion, then at 5, 30, and 55 min during perfusion, and finally at 5 and 60 min and 6 and 24 h after perfusion) and in recirculating perfusate (5, 30, and 55 min). While markedly elevated MMC concentrations (maximum 6290 ng/mL) were found in the liver perfusate, systemic concentrations remained low (maximum 45 ng/mL), indicating no considerable leakage. MMC concentrations in the perfusate constantly decreased during perfusion. After rinsing with 1500 mL saline, a mean concentration of 52.5+/-33 ng MMC/mL was measured in the washout from 5 patients. In 1 patient with a colorectal carcinoma, MMC concentrations in the perfusion medium were 10-fold and in the plasma 2-fold higher than in the other patients. This high MMC concentration caused severe intrahepatic vascular damage and finally led to the patient's death. In conclusion, IHLP and intrahepatic perfusion with MMC resulted in a high response of hepatic tumors. Systemic exposure of MMC can be reduced effectively by isolated perfusion. However, hepatic toxicity of MMC must be considered.

Cigarette smoking stimulates cysteinyl leukotriene production in man.

Chronic cigarette smoking is an important risk factor for pulmonary and cardiovascular diseases. An increased production of cysteinyl leukotrienes has been shown in asthma and in cardiac ischaemia. The effect of cigarette smoking on cysteinyl leukotriene biosynthesis is, however, not known. Urinary leukotriene E4 (LTE4) was measured in 30 habitual smokers and in 30 non-smokers. In a further 12 non-smokers urinary LTE4 excretion was assessed before and after smoking six cigarettes. In addition, the effect of transdermal nicotine on urinary LTE4 excretion was studied in seven non-smokers. There was a close correlation (r = 0.92, P < 0.0001) between urinary excretion of LTE4 and the number of cigarettes smoked daily by habitual smokers. Smoking six cigarettes within 12h resulted in a significant (P = 0.0047), twofold increase in the mean individual LTE4 excretion in non-smokers. Transdermal nicotine had no effect on LTE4 excretion in non-smokers. In conclusion, cigarette smoke causes a dose-related increase in cysteinyl leukotriene production in habitual smokers. Some of the adverse effects of smoking may be related to an enhanced leukotriene synthesis.

Urinary nitrate excretion is increased in patients with rheumatoid arthritis and reduced by prednisolone.

OBJECTIVES: To determine daily production of nitric oxide (NO) measured as urinary nitrate excretion, and the effect of prednisolone in patients with rheumatoid arthritis (RA). METHODS: Twenty four hour urinary nitrate was measured by gas chromatography in 10 patients with RA, before and two to four weeks after commencement of prednisolone 0.5 mg/kg body weight, and in 18 healthy controls. RESULTS: Before the start of prednisolone treatment the urinary nitrate excretion in patients with RA was 2.7-fold greater (p < 0.001) than that in healthy volunteers. After prednisolone it decreased significantly, by 28%, at which time inflammatory activity (as indicated by C reactive protein, erythrocyte sedimentation rate, joint count, and early morning stiffness) was also reduced considerably. Despite this decrease, the urinary nitrate excretion in patients with RA remained twice that in the control group (p < 0.05). CONCLUSIONS: Our data suggest that the endogenous production of NO is enhanced in patients with RA. Furthermore, the results indicate that, in parallel with suppression of inflammation, this increased NO synthesis could be reduced by prednisolone treatment.

Chemical synthesis of dioxygen-18 labelled omega-/beta-oxidized cysteinyl leukotrienes: analysis by gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry.

Cysteinyl leukotrienes (LT) C4, LTD4 and LTE4 are potent mediators of anaphylaxis and inflammation. LTE4 is extensively metabolized in man mainly by omega-oxidation followed by subsequent beta-oxidation to more polar and biologically inactive metabolites. This paper describes a method for the synthesis of [1,20-18O2]-carboxy-LTE4, [1,18-18O2]-carboxy-dinor-LTE4, and [1,16-18O2]-carboxy-14,15-dihydro-tetranor-LTE4 starting from the unlabelled dimethyl esters of 20-carboxy-LTA4, 18-carboxy-dinor-LTA4 and 16-carboxy-14,15-dihydro-tetranor-LTA4, respectively, by separate chemical conjugation with cysteine hydrochloride in H2-18O-methanol followed by alkaline hydrolysis with Li18OH. The isotopic purity of the isolated reaction products was 94% at 18O for all three preparations while only 0.3% remained unlabelled as confirmed by negative-ion chemical-ionization gas chromatography-mass spectrometry (GC-NICI-MS) after their catalytical reduction/desulphurization and derivatization. The 18O2-labelled compounds are demonstrated to be suitable internal standards for quantification by GC-NICI-MS and GC-NICI-tandem MS. We found by GC-NICI-tandem MS that the excretion rate of 20-carboxy-LTE4 is comparable to that of LTE4 (both in nmol/mol creatinine, mean +/- S.E.) in healthy children (26.7 +/- 4.7 vs. 32.0 +/- 6.0, n = 9) and adults (13.9 +/- 1.1 vs. 27.2 +/- 5.4, n = 3).

Neuropeptide levels early after trauma: immunomodulatory effects?

The levels of endogenous opioids, beta-endorphin and methionine-enkephalin, were analyzed in 21 severely traumatized patients (ISS 32, mortality 42.8%) from a first blood sample drawn at the scene of the injury before resuscitation within 32 +/- 16 minutes after the injury and for 8 days after trauma. Additionally, the respiratory burst function of polymorphonuclear neutrophils (PMNs) was assessed and the results were compared with those obtained from 5 healthy control patients undergoing elective surgery with the same analgesic regimen as the multiple trauma patients. Compared with elective surgery anesthesia (controls 3.3, surgery 3.2 fmol/L), the beta-endorphin levels on-scene were markedly elevated (survivors 10.1 fmol/L, non-survivors 15.0 fmol/L) (p < 0.05). Methionine-enkephalin levels after trauma were not different from those of the controls. The stimulation of PMNs with different concentrations of the opioids at the first day after trauma gave results comparable with those of the controls. On the third day after trauma the reactivity of PMNs to low opioid concentrations was markedly suppressed to 79.6% of the baseline value (p < 0.05). Endogenous opioids seem to be able to modulate the nonspecific immune-response after trauma.

Impaired degradation of prostaglandins and thromboxane in Zellweger syndrome.

Cyclooxygenase products are metabolized by omega-oxidation as well as beta-oxidation. Children with Zellweger syndrome (ZS) are characterized by peroxisome deficiency. To evaluate the role of peroxisomal beta-oxidation on cyclooxygenase metabolites, the degradation of endogenous prostaglandin (PG) E2, prostacyclin, and thromboxane (Tx) A2 was assessed in children with ZS (n = 7) and in healthy children (n = 7). PGE2, prostacyclin, TxB2, and their major urinary metabolites 7 alpha-hydroxy-5,11-dioxo-tetranor-prosta-1,16-dioic acid, 2,3-dinor-6-oxo-PGF1 alpha, and 2,3-dinor-TxB2, respectively, were measured in urine by gas chromatography-mass spectrometry/mass spectrometry. The median excretion of healthy children was 17.9 ng of 7 alpha-hydroxy-5,11-dioxo-tetranor-prosta-1,16-dioic acid/mg creatinine (interquartile range, 6.3 to 19.4 ng/mg), 0.38 ng of 2,3-dinor-6-oxo-PGF1 alpha/mg creatinine (interquartile range, 0.34 to 0.70 ng/mg), and 0.36 ng of 2,3-dinor-TxB2/mg creatinine (interquartile range, 0.14 to 0.54 ng/mg). In contrast, none of these metabolites could be detected in urine of children with ZS (p < 0.002). However, we identified in the urine of these children a new metabolite of PGE2 as 11-hydroxy-9,15-dioxo-prost-5-en-1,20-dioic acid by gas chromatography-mass spectrometry, and we confirmed the presence of 9,11-dihydroxy-15-oxo-prost-5-en-1,20-dioic acid the main urinary metabolite of PGF2 alpha in ZS. Importantly, these two metabolites were only detectable in urine of children with ZS.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of cysteinyl leukotriene synthesis in man.

We have developed methods to measure LTE4, the index metabolite of cysteinyl leukotrienes, by HPLC/RIA and GC/MS. Urinary LTE4 appears to be a reliable parameter to assess whole body cysteinyl leukotriene synthesis in man from all evidence available at this time. Measurement of this metabolite can be used to monitor effects of diseases and drugs on cysteinyl leukotriene synthesis in man.

Enhanced synthesis of cysteinyl leukotrienes in juvenile rheumatoid arthritis.

OBJECTIVE: Endogenous synthesis of cysteinyl leukotrienes in juvenile rheumatoid arthritis (JRA) was investigated. METHODS: Cysteinyl leukotriene synthesis was assessed by measuring the excretion of leukotriene E4 (LTE4) in urine by radioimmunoassay. The identity of urinary LTE4 was investigated by gas chromatography-mass spectrometry (GC-MS), and 2,3-dinor-thromboxane B2 was measured with GC-MS. RESULTS: Excretion of LTE4 into urine was significantly (P < 0.05) enhanced in children with JRA compared with that in healthy children (n = 10). Aspirin, in a dosage of 2.5 gm/day, had no effect on urinary LTE4 levels, but it reduced urinary 2,3-dinorthromboxane B2 levels by more than 85% in healthy adults. There was a positive correlation between LTE4 excretion and the number of affected joints. CONCLUSION: This study demonstrates a markedly enhanced cysteinyl leukotriene synthesis and a positive correlation between LTE4 excretion and the number of affected joints in children with JRA.

Gas chromatographic-mass spectrometric determination of leukotriene E4 in human urine using deuterium-labelled leukotriene E4 standards.

The utility of two deuterium-labelled leukotriene (LT) E4 analogs, e.g. [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4, as internal standards for the determination of LTE4 in human urine by gas chromatography-mass spectrometry (GC-MS) was investigated. 2H-Exchange during hydrogenation occurred both in [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4 in an extent of 9.4 +/- 0.5% and 67.3 +/- 0.6% (mean +/- S.D., n = 6), respectively. The lower extent of 2H-exchange in [20,20,20-2H3]LTE4 allowed a more accurate quantitation than the use of [14,15,17,17,18,18-2H6]LTE4. Applying [20,20,20-2H3]LTE4 as internal standard the coefficients of variation for the intra- and inter-assay determination of LTE4 in human urine were 5.7% and 6.2% (n = 4), respectively. The inter-assay coefficient of variation for [14,15,17,17,18,18-2H6]LTE4 was 15%. Using [20,20,20-2H3]LTE4 as internal standard and GC-MS, healthy volunteers were found to excrete 17 +/- 10 nmol LTE4 per mol creatinine (mean +/- S.D., n = 11). Similar excretion rates for LTE4 in urine of healthy volunteers were found using GC-tandem MS with [1,1-18O2]LTE4 as internal standard. Our results demonstrate that [20,20,20(-2)H3]LTE4 is a suitable internal standard for the GC-MS determination of urinary LTE4.

Analysis of leukotrienes, prostaglandins, thromboxane B2 and their metabolites by capillary isotachophoresis.

An analytical capillary isotachophoretic method for the analysis of the eicosanoids leukotriene E4, leukotriene B4, prostaglandins E1 and E2, 6-keto-prostaglandin F1 alpha, thromboxane B2, and their metabolites of omega- and/or beta-oxidation is described. The method is based on anionic separation and detection by UV absorbance (254 nm) and conductivity and allows simultaneous analysis of the primary compounds and their corresponding major urinary metabolites. The method was applicable to the qualitative and quantitative analysis of prostaglandin E1 in a drug preparation.

Enhanced synthesis of cysteinyl leukotrienes in atopic dermatitis.

Synthesis of cysteinyl leukotrienes was assessed in patients with atopic dermatitis (AD; n = 8) and healthy volunteers (n = 8) by measuring urinary excretion of leukotriene E4 (LTE4), the main index metabolite of cysteinyl leukotrienes in man. Using this non-invasive method we demonstrated a significant (P < 0.05) 4.5-fold increase in excretion of LTE4 compared with healthy volunteers. The identity of LTE4 was unequivocally demonstrated by gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS). LTE4 was routinely measured by radioimmunoassay (RIA), and quantitative measurement of LTE4 by RIA was validated by GC-MS/MS. There was a linear correlation between LTE4 measured by RIA and by GC-MS/MS (r = 0.994). In representative samples, LTE4 was also quantitatively assessed by GC-MS/MS. In these samples, LTE4 values obtained by GC-MS/MS differed < 10% from those obtained by RIA. The present findings suggest that cysteinyl leukotrienes play a role in AD.

Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders.

The degradation of leukotrienes by beta-oxidation from the omega-end proceeds in peroxisomes (Jedlitschky et al. J. Biol. Chem. 1991. 266:24763-24772). Peroxisomal degradation of leukotrienes was studied in humans by analyses of endogenous leukotrienes in urines from eight patients with biochemically established peroxisome deficiency disorder and eight age- and sex-matched healthy infant controls. Leukotriene metabolites were separated by high-performance liquid chromatography, quantified by radioimmunoassays, and identified as well as quantified by gas chromatography-mass spectrometry. Urinary leukotriene E4 (LTE4) and N-acetyl-LTE4 excretions, relative to creatinine, were increased > 10-fold in the patients in comparison to healthy infants. The beta-oxidation product omega-carboxy-tetranor-LTE3 averaged 0.05 mumol/mol creatinine in the controls but was not detectable in the patients. However, omega-carboxy-LTE4 (median 13.6 mumol/mol creatinine) was significantly increased in the patients' urine, whereas LTB4 (median 0.07 mumol/mol creatinine) and omega-carboxy-LTB4 were detected exclusively in the urines of the patients. These data indicate an impairment of the inactivation and degradation of both LTE4 and LTB4 in patients with peroxisomal deficiency. The increased levels of the biologically active, proinflammatory mediators LTE4 and LTB4 might be of pathophysiological significance in peroxisome deficiency disorders. This is the first and so far only condition with a pronounced urinary excretion of omega-carboxy-LTE4, omega-carboxy-LTB4, and LTB4. This impaired catabolism of leukotrienes and the altered pattern of metabolites may be of diagnostic value. These findings underline the essential role of peroxisomes in the catabolism of leukotrienes in humans.

The pharmacokinetics of clonidine in high dosage.

We have evaluated the pharmacokinetics of high doses of clonidine, as used in the prophylactic treatment of alcohol withdrawal syndrome, in 11 alcohol-dependent patients undergoing surgery for oesophagogastrectomy. Clonidine was given in a bolus of 150 micrograms followed by a continuous infusion. After a mean period of treatment of 9.2 (range 3 to 26) days and a mean dose of 0.72 (range 0.29 to 2.37) mg per day of clonidine the mean terminal half-life was 15.8 (range 9.9 to 23) h (n = 7). In order to compare initial and terminal half-lives of clonidine intraindividually, four patients were given a bolus of 150 micrograms followed 24 h later by a continuous infusion. The pharmacokinetics of clonidine were described by two exponentials, with a distribution half-life of 1.2 h and a terminal half-life of 14.6 h. After a mean period of 8.3 (range 2 to 15) days and a mean dose of 0.62 (range 0.15 to 1.82) mg per day the terminal half-life in these four patients was 15.6 (range 14.0 to 17.9) h. The relation between dosage and plasma concentration was linear.

Enhanced synthesis of cysteinyl leukotrienes in psoriasis.

Cysteinyl leukotriene synthesis was investigated in patients with psoriasis. A non-invasive test requiring no stimulation was employed by measuring the major index metabolite of LTC4, which appears in urine. The presence of this metabolite, LTE4, was shown unequivocally by gas chromatography-mass spectrometry. Routinely LTE4 was quantitated by specific radio immunoassay after its isolation by reversed-phase high-performance liquid chromatography. Furthermore, in representative samples amounts of LTE4 obtained by radioimmunoassay were validated by gas chromatography-mass spectrometry. We demonstrate a significant (p less than 0.01) more than fourfold increase of urinary LTE4 in psoriasis compared to healthy volunteers. Urinary LTE4 was log normally distributed with geometric mean values (95% confidence intervals) of 11 (9-14) nmol LTE4/mol creatinine in healthy volunteers (n = 11) and 51 (28-95) nmol LTE4/mol creatinine in psoriasis (n = 9). The present study shows that cysteinyl leukotriene synthesis is enhanced in patients with psoriasis and that measurement of urinary LTE4 is a useful parameter to monitor its rate of synthesis.

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography-mass spectrometry.

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E4 (LTE4). Oxygen-18 labelling of LTE4 methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE4 to the extent of 89.8% ([18O2]LTE4) and one oxygen-18 atom to the extent of 9.4% ([16O18O]LTE4), with only 0.7% remaining unchanged ([16O2]LTE4). [18O2]LTE4 was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4 degrees C for up to 20 h. [18O2]LTE4 was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE4 in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE4 in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.