Günther Lamprecht

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the ranking order being: Brussels sprouts > white cabbage > cauliflower > green cabbage > kohlrabi > broccoli > turnip > black radish. In experiments with mammalian cells, six juices induced structural chromosome aberrations. Brussels sprouts, white and green cabbage caused the strongest effects (800 microliters of juice induced a 5-fold increase over the background). In sister chromatid exchange assays, positive results were measured as well, but the effects were less pronounced. With all juices the genotoxic effects seen in mammalian cells were paralleled by a pronounced decrease in cell viability. Column fractionation experiments showed that 70-80% of the total genotoxic activity of the juices is found in the fraction which contains isothiocyanates and other breakdown products of glucosinolates, whereas phenolics and flavonoids contributed to a lesser extent to the overall effects. On the basis of these findings, and considering the negative results obtained with non-cruciferous vegetables (tomato, carrot and green pepper), it seems likely that the genotoxic effects of the juices are due to specific constituents of cruciferous plants such as glucosinolates and/or their breakdown products, in particular, isothiocyanates, which we found previously to be potent genotoxins in bacterial and mammalian cells. Finally, spices (mustards and horse radish paste) and phytopharmaceutical preparations were tested in bacterial assays. Mustards and horse radish caused very weak effects while most of the pharmaceutical preparations gave negative results, except cabbage tablets, which caused a strong and dose dependent induction of his revertants in Salmonella TA100. The present findings clearly indicate that cruciferous vegetables contain DNA damaging constituents. These observations are in contrast to earlier findings, which emphasized the antimutagenic effects of vegetable juices and also raise the question whether greatly increased consumption of Brassica vegetables or their concentrated constituents as a means for cancer prevention is indeed recommendable.

Enantioselective analysis of (R)- and (S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup.

An HPLC column-switching method for the enantioselective determination of (R,S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile-methanol-acetic acid-triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of (R)- and (S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.

Ultra-trace analysis of phenols in water using high-performance liquid chromatography with on-line reaction detection

Abstract An ultra-sensitive HPLC method for the determination of phenols in water based on flow-through reaction is described. The phenols are concentrated by a two-step liquid-liquid extraction with dichloromethane and after evaporation of the organic solvent the separation is carried out on an octadecyl-silica column by a gradient of water-acetonitrile. Detection is performed by an on-line twin detection system. First dinitrophenols are detected by UV-absorption measurement followed by oxidation of phenols with cerium(IV) in a tubular flow-through reactor and fluorescence measurement of cerium(III). Detection limits in the lower ppt range can be achieved. The influence of reaction time, temperature and reagent concentration on reaction yield was investigated. Identification and quantitation is improved by use of internal standards. The method was applied to samples of surface and drinking water.

Different Effects of Exercise on Plasma Concentrations of Nebivolol, Bisoprolol and Carvedilol

AbstractBackground: In-vitro studies have shown that beta-blockers are taken up into and released from adrenergic cells together with epinephrine and norepinephrine. Consequently, studies in humans revealed an increase in plasma concentrations of propranolol and atenolol, whereas those of carvedilol were not affected by physical exercise. However, nebivolol and bisoprolol never were investigated on this issue. Methods: Ten healthy males received oral doses of 5 mg nebivolol, 5 mg bisoprolol, and 50 mg carvedilol daily for one week in a cross-over fashion. Exercise was performed at 3 hours following oral intake of the respective last drugs on the eighth day. Blood samples were taken at rest, during the last minute of exercise, and after 15 min of recovery. Results: At rest and during exercise, heart rates were as follows: Nebivolol, 57 ± 7 and 137 ± 11 beats/min; bisoprolol, 55 ± 5 and 139 ± 14 beats/min; carvedilol, 56 ± 5 and 135 ± 13 beats/min, with no significant differences between the drugs. Plasma concentrations were as follows: Nebivolol—rest 0.273 ± 0.029 ng/ml, exercise 0.274 ± 0.035 ng/ml, recovery 0.272 ± 0.035 ng/ml (n.s.). Bisoprolol—rest 4.99 ± 2.73 ng/ml, exercise 6.49 ± 5.58 ng/ml, recovery 4.90 ± 3.06 ng/ml (p < 0.01). Carvedilol—rest 10.3 ± 9.3 ng/ml, exercise 9.7 ± 8.2 ng/ml, recovery 6.5 ± 5.6 ng/ml (p < 0.05). Discussion: Plasma concentrations of bisoprolol increased during exercise and returned to baseline during recovery, a behaviour which would have been predicted according to present knowledge. However, exercise had no effect on plasma concentrations of nebivolol and carvedilol, a finding that is in contrast to previous results with other beta-blockers such as propranolol and atenolol. We conclude that both nebivolol and carvedilol are not taken up into and released from adrenergic nerves during exercise, a feature that clearly distinguishes these drugs from other beta-blockers so far investigated.

Enantioselective analysis of (R)- and (S)-carvedilol in human plasma by high-performance liquid chromatography

SummaryAn HPLC column-switching method has been developed and validated for the enantioselective determination of (R)- and (S)-carvedilol in human plasma. Sample preparation was performed either off-line, by extraction with trichloromethane and back-extraction into 0.01m aqueous citric acid which was injected on to a LiChrosorb RP 8 column, or on-line, by injecting diluted (0.1m formic acid) plasma on to a LiChrosorb ADS column. In both instances separation was performed by gradient elution and on-line transfer of the fraction containing, the carvedilol on to an enantioselective Teicoplanin column. The enantiomers of carvedilol were separated isocratically by use of methanol-acetonitrile-triethylammonium acetate, 70:30:0.05 (v/v/w), as mobile phase. With fluorescence detection the limits of quantitation were 0.30 ng mL−1 for (R)-carvedilol and 0.26 ng mL−1 for (S)-carvedilol; these were sufficient to enable investigation of the effect of exercise on plasma concentrations of (R)- and (S)-carvedilol after oral administration of either the racemate or the pure enantiomers.Although the operating conditions were optimized for sample preparation by on-line deproteination on a LiChrospher RP 18 ADS column, the complete method was insufficiently rugged for analysis of large numbers of plasma samples—the enantioselectivity of the Teicoplanin column deteriorated too rapidly because of the transfer of enantioselectivity-poisoning interferences which could not be suppressed sufficiently. In contrast the liquid-liquid sample-extraction procedure combined with column switching resulted in a analytical method with long-term stability.

Detection of mutagenic activity in textiles with Salmonella typhimurium

A hundred and ninety-six textile samples were tested in a modified version of the Salmonella/microsome assay for release of mutagenic contaminants. As heat sterilization of the samples can result in reduction of mutagenic activity, tests were performed with streptomycin resistant derivatives of Salmonella tester strains TA98 and TA100. Textile samples were preincubated in buffered saline (PBS), DMSO or ethanol. Subsequently, the fabrics were placed on streptomycin supplemented selective agar plates. In total, 18 samples (9.2%) exerted mutagenic activity. DMSO was the most effective solvent (15 positives) followed by ethanol (9 positive samples) and PBS (7 positives). Most fabrics (16) caused mutagenic effects only upon metabolic activation with liver S9 mix. Chemical analysis indicates that the positive results obtained with PBS are not due to release of histidine or formaldehyde. Three directly active samples gave negative results in strain TA98NR which is devoid of classical nitroreductase. With one exception all other textiles were negative in strain TA98/1,8-DNP6 (which lacks O-acetyltransferase). These findings indicate that nitroaromatics and amines might be responsible for the mutagenic effects of the textiles.

Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using three retention mechanisms

An automated two-dimensional HPLC method for the determination of neopterin in serum is described. Neopterin is separated from proteins on a short octadecylsilica column by size exclusion and from the majority of the other serum components by adsorption. The fraction containing neopterin is transferred by column switching to a solvent-generated cation-exchange column using dodecylsulfonic acid as surface activator. Parameters influencing the separation performance and sensitivity of the fluorescence detection are discussed. The efficiency of the cleaning of the first column was optimized. The method was validated. It achieves a precision of 1% (R.S.D.) and a detection limit of about 0.3 nmol/l. The accuracy is nearly 100%. The method allows a high sample throughput, requiring 15 min per sample.

Exercise does not Affect Plasma Concentrations of (R)- and (S)-Carvedilol

AbstractPurpose: In vitro studies have shown that beta-blockers are taken up into and released from adrenergic cells. As a consequence, plasma concentrations of beta-blockers increase during exercise together with those of epinephrine and norepinephrine. However, effects of exercise on plasma concentrations of (R)- and (S)-carvedilol are unknown. Methods: Twelve healthy males received oral single doses of 12.5 mg (R)-carvedilol, 12.5 mg (S)-carvedilol and 25 mg (R,S)-carvedilol in a cross-over fashion; 11 patients with essential arterial hypertension were given 25 mg (R,S)-carvedilol. Exercise was performed 3 hours following drug intake, and blood samples were taken at rest, at the end of exercise, and after 15 min of recovery. Plasma concentrations of (R)- and (S)-carvedilol were determined by HPLC. Results: Plasma concentrations of (R)-carvedilol were 2- to 3-fold higher than those of (S)-carvedilol (p < 0.05 in all cases). Plasma concentrations of both (R)- and (S)-carvedilol remained unaffected during exercise and recovery. Conclusions: Contrary to all other beta-blockers so far investigated, exercise had no effect on plasma concentrations of (R)- and (S)-carvedilol. We conclude that neither (R)- nor (S)-carvedilol is released from adrenergic cells during exercise, a feature that clearly distinguishes carvedilol from other beta-blockers. Thus, the human organism appears to handle (R)- and (S)-carvedilol differently than other beta-adrenoceptor antagonists. This finding deserves further investigation on a molecular and cellular level in order to clarify these differences between the pharmacokinetics of carvedilol and other beta-blockers.

Direct determination of benzo[a]pyrene in oil distillates by on-line two-dimensional HPLC with column switching

SummaryA selective and sensitive HPLC method for the determination of Benzo[a]pyrene (B[a]p) in oil fractions by means of column switching is described. The diluted oil samples were injected directly onto a silica column with isooctane as eluent. After fast elution of the main part of the sample matrix, the B[a]p containing fraction was transferred on-line to a dinitro-aryl-modified silica column for final separation with isooctane/tetrahydrofuran. A detection limit of 50 ppt B[a]p was found when using fluorescence detection.

Assay of S-ethyl-N-acetyl-l-cysteine in urine by high-performance liquid chromatography using post-column reaction detection

The assay of the ethyl chloride metabolite S-ethyl-N-acetyl-L-cysteine in human urine by HPLC is described. The compound is enriched by adsorption on a non-polar adsorbent of graphitized non-porous carbon, and then stripped from positively charged compounds by application onto a strong acid cation-exchanger. Subsequently, an enzymatic deacetylation is carried out and the acylase is removed by centrifugal ultrafiltration. Separation of the sample is performed by cation-exchange chromatography applying an eluent of a very low elution strength (diluted formic acid). In the column effluent S-ethyl-L-cysteine is derivatized by o-phthaldialdehyde and the reaction product is detected by fluorescence measurement. In human urine a detection limit in the low ppb range is achieved.