International collaborative assessment study of the AHD575 method for the measurement of blood haemoglobin.

Accurate, economical methods for haemoglobin determination by laboratories in countries with limited resources are not available. This report provides the results of an international collaborative study evaluating the alkaline haematin detergent (AHD575) method as a reference method for laboratory services with limited resources. The study included 6 laboratories; 3 in East Mediterranean countries, 1 in East Africa and 3 in Europe. The (AHD575) method was evaluated against the HiCN method, with blood samples drawn from healthy and sick subjects. The results indicate that the AHD575 method is suitable for measuring haemoglobin in laboratories at all levels.

International collaborative assessment study of the AHD [575] method for the measurement of blood haemoglobin

Accurate, economical methods for haemoglobin determination by laboratories in countries with limited resources are not available. This report provides the results of an international collaborative study evaluating the alkaline haematin detergent [AHD[575]] method as a reference method for laboratory services with limited resources. The study included 6 laboratories; 3 in East Mediterranean countries, 1 in East Africa and 3 in Europe. The [AHD[575]] method was evaluated against the HiCN method, with blood samples drawn from healthy and sick subjects. The results indicate that the AHD[575] method is suitable for measuring haemoglobin in laboratories at all levels

Development of a method for sample preparation for subsequent identification and measurement of 1,2,3,4-tetrahydroisoquinolines and other potentially neurotoxic compounds by high-performance liquid chromatography with ultraviolet and fluorescence detection in blood plasma of Parkinson's disease patients.

We developed sample preparation methods for the detection of various biogenic phenylethylamine derivatives such as 3,4-dihydroxyphenylalanine, and their cyclisation products with aldehydes, i.e., 1,2,3,4-tetrahydroisoquinoline derivatives in blood samples. 1,2,3,4-Tetrahydroisoquinolines are considered to play an essential role as neurotoxic compounds in the pathomechanism of Parkinson's disease. We used reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection for separation and identification. Ultrafiltration, protein precipitation and solid-phase extraction were investigated for purification of blood samples and enrichment of various compounds with a wide range of hydrophilicity and hydrophobicity. Protein precipitation by methanol and perchloric acid is a fast method to separate the analytes from the plasma matrix. A higher yield of the analytes is attained with prior addition of an alkylsulfonic acid giving a fine-grained precipitate. With the addition of ion pairing compounds into the sample it is possible to enrich not only lipophilic compounds such as norharman, tryptamine and melatonin, but also hydrophilic ones such as 3,4-dihydroxyphenylalanine by reversed-phase solid-phase extraction. Ultrafiltration is not useful as a screening method.

High-performance liquid chromatographic separation and measurement of various biogenic compounds possibly involved in the pathomechanism of Parkinson's disease.

The work presented here describes an optimised, reversed-phase high-performance liquid chromatographic (RP-HPLC) method for separating 46 biogenic compounds, which, as neurotoxins or as their precursors or derivatives, may be relevant in the pathomechanism of Parkinson's disease. In some cases, the physico-chemical properties of these substances are very similar, in other cases they differ greatly. In order to facilitate their detection in one chromatographic run, ion-pair chromatography was uniquely combined with a gradient elution. A diode array or a dual wavelength detector was used in combination with a fluorescence detector to verify the identity of the compounds.

Detection of DNA-crosslinking agents with the alkaline comet assay.

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37 degrees C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed.

Genotoxic evaluation of three heterocyclic N-methylcarbamate pesticides using the mouse bone marrow micronucleus assay and the Saccharomyces cerevisiae strains D7 and D61.M.

The carbamate insecticides benfuracarb, carbosulfan and furathiocarb were investigated in the mouse bone marrow micronucleus assay to establish whether they show cytogenetic activity in vivo. Two doses of each substance were administered intraperitoneally to NMRI mice. All of the three substances led to a positive micronucleus response in polychromatic erythrocytes of the bone marrow at different expression times. While furathiocarb and carbosulfan showed similar patterns of the time-dependence of the micronucleus formation with maximum values after 72 h, benfuracarb exhibited a different behaviour with the maximum increase taking place within 24 h after substance application. In furathiocarb-treated animals the ratio of normochromatic to polychromatic erythrocytes showed a dose and time depending increase with the highest value obtained after 72 h in animals treated with the upper dose. The two yeast test systems Saccharomyces cerevisiae strains D7 and D61.M were applied in order to evaluate the genetic endpoints gene mutation, gene conversion and aneuploidy induction. None of the three insecticides had an influence on the frequencies of gene conversion and reverse mutation in the yeast S. cerevisiae D7 when tested with and without metabolic activation. In strain D61.M however benfuracarb and furathiocarb led to an increase of chromosome loss in the presence of the S9 metabolizing system.

Effects of benzofuran and seven benzofuran derivatives including four carbamate insecticides in the in vitro porcine brain tubulin assembly assay and description of a new approach for the evaluation of the test data.

The influence of benzofuran and 7 benzofuran derivatives, including the carbamate insecticides benfuracarb, carbofuran, carbosulfan, and furathiocarb, on the in vitro assembly kinetics of porcine brain tubulin was investigated. A new approach to the evaluation of the raw data was made based on polynomial regression and the calculation of a polynomial function of the 11th degree fitting the raw data. By this procedure it is possible to calculate the parameters defining the shape of the absorbance curves and more parameters than those used so far can be included in the analysis of substance effects. In detail, the following curve parameters of the dependence of optical absorption on time were included in the evaluation of the substances of interest: the difference between maximum and minimum absorbance as a measure for the polymerization degree, the coordinates of the turning point of the curve, the slope of the tangent at the turning point which represents the maximum reaction velocity, the mean slope between the points with 10% absorbance increase and 90% absorbance increase and the duration of the lag phase. Out of the eight compounds tested, only the carbamate insecticides had distinct effects on the in vitro polymerization of tubulin, whereas benzofuran and the three 2,3-dihydro-2,2-dimethylbenzofuran derivatives without a carbamate function were inactive. Benfuracarb, carbofuran, carbosulfan, and furathiocarb led to a dose-dependent reduction of the polymerization degree of tubulin as well as to reduction of the maximum and mean reaction velocities. The strongest effects were obtained with furathiocarb and benfuracarb.

Investigation of genotoxic effects of the anti-asthmatic and anti-inflammatory drugs Apocynin and Acetosyringenin in the Salmonella typhimurium mutagenicity assay and the SCE-test with human lymphocytes.

We examined the possible genotoxicity of the recently isolated or developed anti-asthmatic and anti-inflammatory substances Apocynin (CAS 498-02-2) and Acetosyringenin (CAS 2478-38-8) using short term test systems. Apocynin is a constituent of Picrorhiza kurroa, a plant from the Himalaya region. Acetosyringenin is its methoxylated synthetic derivative. The test systems used were the Salmonella typhimurium mutagenicity assay (Ames test) with the tester strains TA 97, TA 98, TA 100, and TA 102, and the sister chromatid exchange assay (SCE-Test) performed with human peripheral lymphocytes. The genetic endpoints covered by these test systems are the induction of gene mutations and the induction of SCEs, which is interpreted as an indicator for DNA-damaging properties of the test compound. The results obtained with Apocynin and Acetosyringenin do not demonstrate genotoxic activities of these compounds regarding the investigated endpoints.

The Ca(2+)-transport-ATPase of human erythrocytes as an in vitro toxicity test system--acute effects of some chlorinated compounds.

Investigations to determine the inhibitory activity on the Ca(2+)-transport-ATPase of human erythrocyte membranes were performed with various compounds of toxicological significance, mostly chlorinated and mainly used as biocides, such as phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,4-dichlorophenol, 2,3,4-trichlorophenol, 2,3,4,5-tetrachlorophenol, pentachlorophenol (PCP), captan, folpet, captafol, (+)-camphene, toxaphene, dichlorodiphenyltrichloroethane (DDT), lindane, endrin, dieldrin, alpha-endosulfan, beta-endosulfan, paraquat, diallate, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Some of the compounds investigated display an inhibitory effect on the Ca(2+)-transport-ATPase at very low concentrations. The in vitro results obtained in this enzyme assay can be correlated directly with the results of other in vitro assays and with the results of in vivo investigations in different species in which an inhibitory effect on various biological functions is observed. Therefore, an inhibitory effect on the Ca(2+)-transport-ATPase indicates a toxic effect of these compounds to cell functions. Since the inhibitory effect of these compounds can be measured rapidly and the enzyme is easy to handle, it might be a useful tool to screen the toxic effects of various compounds on cell function. The aim of the authors was to investigate the usefulness of this screening test system for the characterization of the cellular toxicity of various compounds.

Preparation, purification and characterization of chlorohaemin.

Preparation of chlorohaemin (CAS 16009-13-5) is performed on the basis of the method of Labbe and Nishida (acetone-acetic acid-SrCl2 method) with some significant modifications. Instead of blood as starting material, a fresh preparation of purified human erythrocytes is used. This avoids any contamination with serum and erythrocyte proteins and lipids of the end product. Special care is taken to remove contaminating globin by introducing some specific purification steps during isolation and recrystallisation. The yield is in the range 65-75% of theory, and the purity of the product is better than 99.9% as shown by elemental analysis and specific tests on various compounds as possible contaminants which originate from the starting material such as lipids and proteins and/or from the different steps of preparation and purification during the procedure. The pure chlorohaemin which is the compound of choice as reference substance for the AHD method in haemoglobinometry is characterized by LSI mass spectrometry (m/z = 616, haemin ion), field desorption-mass spectrometry (m/z = 652), by IR-spectroscopy, and by UV/VIS absorption spectroscopy (pyridine haemochrome spectrum, AHD spectrum).

Development of a method to increase the proportion of polychromatic erythrocytes from mouse bone marrow for more rapid evaluation of the micronucleus assay. Comparison with the conventional method with respect to micronucleus frequency and time required for preparation and evaluation.

By treatment of bone marrow suspension in hypotonic (0.51%) saline, followed by purification on a cellulose column, the proportion of polychromatic erythrocytes in bone marrow smears can be increased 2.6-fold. Furthermore, practically all nucleated cells of erythropoietic or leukopoietic origin are removed. This kind of bone marrow preparation can lead to a considerable reduction in the time required for microscopical evaluation of the micronucleus assay without altering the rate of micronucleated polychromatic erythrocytes in a negative (isotonic saline) and a positive (mitomycin C) control group.

The determination of haemoglobin as cyanhaemiglobin or as alkaline haematin D-575. Comparison of method-related errors.

In order to compare the accuracy of haemoglobin (Hb) determination methods, the commonly used cyanhaemiglobin (HiCN) method and the recently developed alkaline haematin D-575 (AHD) method (R. Zander, W. Lang & H. U. Wolf (1984) Clin. Chim. Acta 136, 83-93; H. U. Wolf, W. Lang & R. Zander (1984) Clin. Chim. Acta 136, 95-104) were tested with respect to method-related errors such as plasma, cell, and Hb errors. Both methods yield a series of more or less significant errors which generally lead to an overestimation of the Hb concentration in the order of 1%. However, in all three cases of plasma errors, i.e. normal plasma error, plasma error in lipaemic blood, and plasma error in bilirubinaemic blood, the AHD method shows significantly lower values of errors than the HiCN method. In the case of cell errors such as ghost and leukocyte errors, the overestimation of the Hb concentration by the HiCN method is 60% higher than that by the AHD method. In the case of Hb errors such as fetal Hb and carboxy Hb errors, there is a significant overestimation of the Hb concentration by the HiCN method, which amounts 3 min after mixing of blood and HiCN solution to 0.7% in the case of fetal Hb and to 13.2% in the case of carboxy Hb. The latter value yields an overestimation of 1.3%, when 10% carboxy Hb in a blood sample is present. In contrast, there is no detectable overestimation after 3 min in the case of the AHD method. Thus, the AHD method provides a higher accuracy in Hb determination than the commonly used HiCN method.

[Tetrahydrocannabinols in hair of hashish smokers]. / Tetrahydrocannabinole im Haar von Haschischrauchern.

The study investigates the presence of tetrahydrocannabinols in the head hair and the pubic and axillary hair. The hair samples were obtained from hashish smokers. The concentrations were determined by radioimmunoassay and reflect total tetrahydrocannabinols and metabolites. The values found ranged from 0.4 ng/mg hair up to 3.8 ng/mg hair. The presence of the drug in the hair samples was also demonstrated by GC/MS.

Detection of methadone in human hair by gas chromatography/mass spectrometry.

Determination of methadone in human hair by gas chromatography/mass spectrometry was described. Helium as carrier gas, a 30-m bonded phase-fused silica DB-1 capillary column and splitless injection at 230 degree C temperature were used. The concentrations of methadone and its metabolites were measured in addition by radioimmunoassay (RIA). Both methods GC/MS and RIA showed the presence of methadone in human hair.

Methadone concentrations in human hair of the head, axillary and pubic hair.

The concentrations of methadone and its metabolites in the hair of the head, axillary and pubic hair obtained from patients receiving a daily maintenance doses, were determined. Comparison of the concentrations provides the highest values in the axillary hair, followed by pubic hair and the hair of the head.

Determination of methadone in human hair by radioimmunoassay.

The concentrations of methadone in human hair were measured. The washed hair was cut in 1-mm pieces approximately, then incubated overnight at 45 degrees C with 0.1 m HCl. The extracts were alcalized by 1 m NaOH and diluted by phosphate buffer, pH 7.4. The methadone concentrations were determined by radioimmunoassay. The method is simple, rapid, and practicable for routine determination.

Genetic effects of chlorinated ethylenes in the yeast Saccharomyces cerevisiae.

The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae. From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates. Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not. The 2 ways of metabolic activation were compared and were found to cause approximately the same effect. In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M. The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss. In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM.

Mutagenic activity of acetonitrile and fumaronitrile in three short term assays with special reference to autoinduction.

Two aliphatic nitriles, acetonitrile and fumaronitrile were tested for their genotoxic potential in three mutagenicity test systems: the Salmonella/microsome-assay, an assay using Saccharomyces cerevisiae (strain D7), and the bone marrow micronucleus test. Both compounds were tested with and without metabolic activation in the yeast and the bacterial test systems using S9 preparations from phenobarbitone-pretreated and autoinduced rats. Autoinduction was performed by chronic (7 days) application of a dose equivalent to a 5% oral LD50-value of the respective compound. With yeast strain D7 both nitriles induced low levels of gene conversion in the presence of phenobarbitone-induced liver homogenate. An increase in the number of ile+-revertants was not detectable under any condition. Neither of the compounds showed mutagenic activity in the Ames test with or without metabolic activation. A weak positive effect of acetonitrile could be detected in the micronucleus test 24 h after i.p.-injection of the compound using a dose of 60% LD50. Fumaronitrile showed positive results with a 50% LD50 dose 48 h after administration to mice not preinduced. After 1 week of autoinduction these effects did not appear anymore, with the exception of acetonitrile 72 h after application of a dose amounting to 60% of the oral LD50-value.