Potentially hallucinogenic 5-hydroxytryptamine receptor ligands bufotenine and dimethyltryptamine in blood and tissues.

Bufotenine and N,N-dimethyltryptamine (DMT) are hallucinogenic dimethylated indolethylamines (DMIAs) formed from serotonin and tryptamine by the enzyme indolethylamine N-methyltransferase (INMT) ubiquitously present in non-neural tissues. In mammals, endogenous bufotenine and DMT have been identified only in human urine. The DMIAs bind effectively to 5HT receptors and their administration causes a variety of autonomic effects, which may reflect their actual physiological function. Endogenous levels of bufotenine and DMT in blood and a number of animal and human tissues were determined using highly sensitive and specific quantitative mass spectrometric techniques. A new finding was the detection of large amounts of bufotenine in stools, which may be an indication of its role in intestinal function. It is suggested that fecal and urinary bufotenine originate from epithelial cells of the intestine and the kidney, respectively, although the possibility of their synthesis by intestinal bacteria cannot be excluded. Only small amounts of the DMIAs were found in somatic or neural tissues and none in blood. This can be explained by rapid catabolism of the DMIAs by mitochondrial monoamino-oxidase or by the fact that the dimethylated products of serotonin and tryptamine are not formed in significant amounts in most mammalian tissues despite the widespread presence of INMT in tissues.

Scaled down method for the reproducible recovery to high purity of human serum albumin from low volume blood samples.

A basic need for a protein-based dosimeter is a purified protein. In this communication we present an isolation protocol and an HPLC-based assay which allows one to determine the purity of the isolated albumin. A total of 168 human blood samples were collected from workers of a benzene processing plant and from nearby countryside at Kohtla-Järve, Estonia. Albumin was isolated from plasma by sequential precipitation and the purity was determined by HPLC. The amount of albumin present in plasma varied between the individuals, being 147 +/- 26 mg/5 ml (n = 168), which is about 59% of plasma albumin. However, the isolated albumin was highly pure (100.9 +/- 8.2%, n = 5). All albumin samples analyzed demonstrate two peaks in HPLC analysis. The two peaks detected were collected and subjected to MS analysis, which demonstrates a difference of 120 mass units between the two albumin products isolated. We have developed an assay, which is easy to carry out and is not too labor intense. The HPLC analysis can be applied to confirm the purity of the isolated albumin as well as to confirm the quantity of the albumin in samples.

Gas chromatographic-mass spectrometric analysis of some potential toxicants amongst volatile compounds emitted during large-scale thermal degradation of poly(acrylonitrile-butadiene-styrene) plastic.

A number of compounds emitted during the thermal degradation of plastics are potentially toxic. This study was aimed at identifying the volatile compounds emitted during large-scale thermal degradation of poly(acrylonitrile-butadiene-styrene). About 5 g of the sample were degraded at between 25 and 470 degrees C in air and nitrogen in a device that can simulate temperature-programmed thermogravimetry. The volatiles were collected in dichloromethane using the solvent trap technique. Some of the 92 compounds identified by gas chromatography-mass spectrometry were found to have no hitherto documented toxicological profiles, even though they are potentially dangerous.

Chlorinated dioxins and dibenzofurans in the environment--a hazard to public health?

Polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF) are globally distributed, are persistent in the environment, and tend to accumulate in human tissues. Several 2,3,7,8-substituted PCDD and PCDF have been found at parts-per-trillion levels in human milk and adipose tissue. Food is the major source for the general population. Above-average exposures may be caused by the incineration of various wastes, certain industries (eg, metal and paper and pulp), and traffic exhaust. PCDD and PCDF have been of great public concern because one of the congeners, 2,3,7,8-tetrachlorodibenzodioxin, is an extremely potent carcinogen and teratogen in rodents. Although epidemiologic studies on cancer are too few for conclusions, animal studies and documented human exposure suggest that humans may be at increased risk. Human exposure is generally low when compared with the effective levels for rodents. However, part of the population can be more exposed and, consequently, also be potentially at higher risk.

The analysis of trichothecenes in wheat and human plasma samples by chemical ionization tandem mass spectrometry.

Ammonia desorption chemical ionization (D/CI) tandem mass spectrometry (method A), isobutane D/CI tandem mass spectrometry using reactive collisions with ammonia (method B), and gas chromatography negative CI (GC-NCI) tandem mass spectrometry (method C) were compared for the detection and quantitation of trichothecenes in spiked human plasma and wheat samples. The trichothecenes were analyzed as their heptafluorobutyrate (HFB) esters in method C and without derivatization using the direct exposure probe in methods A and B. The instrument was operated in the multiple reaction mode. With standard solutions, the detection limits were about two orders of magnitude better with method C (0.1-2 pg) than with methods A and B. The trichothecenes could be detected in the spiked samples at lower concentration levels by method C (0.001 microgram g-1) than by methods A and B (0.01-0.1 microgram g-1), since the sensitivity and selectivity of method C were better than those of methods A and B. Because of the need of derivatization, method C was more time-consuming than methods A and B. Iso-T-2 was used as an internal standard in method C and a steroid nandrolone in methods A and B. The linearities and reproducibilities of the quantitation were better with methods A and C than with method B.

Identification of the phyto-oestrogen 3',7-dihydroxyisoflavan, an isomer of equol, in human urine and cow's milk.

The isoflavonoid diphenol 3',7-dihydroxyisoflavan, an isomer of the known compound equol (4',7-dihydroxyisoflavan), has been identified in human urine and in cow's milk. The compound was isolated as the glucuronide, purified by column chromatography and identified after hydrolysis to the aglycon. The trimethylsilyl ether derivative was characterized by comparison of its mass spectrum and chromatographic properties with those of synthesized silylated isomers of equol.

Detection of DNA adducts by postlabelling with 3H-acetic anhydride.

A method has been developed for detecting 7-alkylguanines which is based on derivatization of hydroxyl and primary amino groups with 3H-acetic anhydride. The derivatization procedure is devised for 7-methylguanine, with examination of reaction kinetics. The method is applied to the detection of the isomeric styrene oxide-guanine adducts; styrene-7,8-oxide is the active metabolite of styrene. The procedure can be used as a postlabelling method for the simultaneous detection of several adducts after selective depurination of 7-alkylguanines from DNA.

Characterization of trichothecenes by ammonia chemical ionization and tandem mass spectrometry.

Ammonia and deuterated ammonia chemical ionization (CI) mass spectra and collisionally activated dissociation (CAD) mass spectra of ammonium adduct ions are presented for ten trichothecenes. The samples were introduced by direct exposure probe. Effects of ion source temperature and pressure on the ammonia CI gas plasma and the formation of the ammonium adduct ion were studied. The CI conditions were optimized to produce a maximal yield for the ammonium adduct ion of trichothecenes, i.e. the parent ion for tandem mass spectral analysis. Besides source temperature and pressure, proton affinity and the stability of the ammonium adduct ion affect the relative abundance ratio of [M + H]+:[M + NH4]+ in ammonia CI and CAD mass spectra. The ratio [M + H]+:[M + NH4]+, and hence the stability of the ammonium adduct ion, are largely determined by the functional groups (hydroxy, carbonyl, acetoxy, and isovaleroyloxy) and their location in the trichothecene nucleus. The most abundant fragment ions in the ammonia CI spectra and the most abundant daughter ions in the CAD spectra of the ammonium adduct ions are formed by the losses of ammonia and functional groups as neutrals in various combinations.

Kinetics of hydrolysis in vitro of nornitrogen mustard, a metabolite of phosphoramide mustard and cyclophosphamide.

A gas chromatographic (GC-ECD) method was developed for the determination of nornitrogen mustard (NOR) and its hydrolysis products. The method was based on derivatization by heptafluorobutyric anhydride. The structures of the derivatives of NOR were established by GC-MS. The method was used to characterize the rate of transformation of NOR and phosphoramide mustard (PAM), important metabolites of cyclophosphamide, into secondary products in vitro at 37 degrees C and pH 7.4. The rate of disappearance of NOR had a half-life of 20 min. The half-life of appearance of N-(2-chloroethyl)-N-(2-hydroxyethyl)amine (NOR-OH) was 19 min. While most NOR appeared to be converted to NOR-OH, the yield of N,N-bis(2-hydroxyethyl)amine (NOR-OH-OH) was a small fraction of the starting material. The disappearance of NOR, when PAM was used as a starting material, had a half-life of 19 min; in these experiments NOR-OH and NOR-OH-OH were relatively much more abundant compared to when NOR was used as the starting material.

Characterization of reaction products between styrene oxide and deoxynucleosides and DNA.

Styrene oxide was reacted with deoxynucleosides and DNA in aqueous buffer at pH 7.4. The products were purified by HPLC, characterized by UV spectroscopy and by chemical ionization mass spectrometry. The main products identified were 7-alkyl-, N2-alkyl- and O6-alkyldeoxyguanosine, 1-alkyl-, and N6-alkyldeoxyadenosine, N4-alkyl-, 3-alkyl- and O2-alkyldeoxycytidine and 3-alkylthymidine. The relative yields of alkylated deoxynucleosides were dG greater than dC greater than dA greater than T. In the reactions of styrene oxide with DNA the dominant product isolated was 7-alkylguanine but N2-alkylguanine was also detected.

Reaction products of styrene oxide with guanosine in aqueous media.

Styrene oxide was reacted with guanosine in fully aqueous conditions, and the products were purified by cation exchange chromatography and h.p.l.c. The products were characterized by u.v. and mass spectra, loss of tritium from the C-8 position and dissociation properties. About 90% of the total product was identified, of which 57, 28 and 15% were assigned to N-7, N2 and O6 adducts. The two diastereomers of beta-substitution accounted for 53% of the N-7 adducts, while the two alpha-substitutions accounted for the remaining 47%. Two species of N2 and O6 products were also isolated but their substitution pattern was not established.

Styrene and styrene oxide concentrations in the air during the lamination process in the reinforced plastics industry.

Styrene and styrene oxide concentrations were measured during the lamination process in the reinforced plastics industry. The mean concentration of styrene in the personal samples was 130 ppm, the highest value measured being 350 ppm. The average concentration for styrene oxide alone was 0.1 ppm, whereas the corresponding measurement for styrene oxide and its decomposition products combined was 0.7 ppm. In comparison then, the concentrations of styrene oxide and its derivatives were much lower (about 0.5% of the total) than those of styrene.

Fetal sulfated and nonsulfated bile acids in intrahepatic cholestasis of pregnancy.

Nonsulfated the sulfated bile acids were determined in maternal and umbilical cord serum, amniotic fluid, and meconium samples in pregnancies complicated by maternal intrahepatic cholestasis, and the results were compared with those obtained in uncomplicated pregnancy. The serum levels of nonsulfated and sulfated bile acids were elevated in both maternal and fetal serum in cholestasis pregnancies, and there was a considerable maternal-to-fetal difference in their levels across the placenta, the lower values being in the fetal compartment. The proportion of sulfate conjugates of the total serum bile acids was similar in both compartments, about 10%. In amniotic fluid low levels of cholic and chenodeoxycholic acid were found in uncomplicated pregnancies. In the cholestasis pregnancies amniotic fluid bile acid levels were elevated, especially that of cholic acid. The swallowing of considerable amounts of bile salt by the fetus with the amniotic fluid leads to an increased accumulation of bile salt in the meconium in cholestasis. The proportion of sulfate-conjugated bile acids was large in the meconium samples in both uncomplicated and cholestasis pregnancies.

Biliary bile acids in uncomplicated pregnancy and in cholestasis of pregnancy.

Conjugated and sulfated bile acids were determined by gas-liquid chromatography and by high-pressure liquid chromatography in gallbladder bile samples of four pregnant women at term and of two patients with cholestasis of pregnancy. In healthy pregnant women the mean ratio of cholyl/chenodeoxycholyl/deoxycholyl glycine was 3.7 : 1 : 0.23 and that of taurine conjugates 3.0 : 1 : 0.25, respectively. In gallbladder bile pool of non-pregnant females these ratios were 1.0 : 1 : 0.00 and 1.0 1 : 0.70, respectively. Thus cholic acid predominated in pregnancy bile. In patients with cholestasis of pregnancy, cholid acid comprised 90% of total biliary bile acids, the proportion of chenodeoxycholic acid was greatly decreased and nonsulfated deoxycholic acid was not detected. The proportion of sulfated bile acids of total biliary bile acids was between 0.4 and 1.2% in uncomplicated pregnancy and 0.3 and 0.5% in cholestasis patients.

Determination of serum bile acids by glass capillary gas-liquid chromatography.

Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gas-liquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxycholic and deoxycholic acids in a group of healthy fasting women was 2.14 mumol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 mumol/l and at 38-41 weeks of gestation 2.10 mumol/l. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 mumol/l.