Chiral gas chromatographic determination of ephedrine-type alkaloids in dietary supplements containing Má Huáng.

Má Huáng is a traditional Chinese medicine derived from the aerial parts of several Ephedra species (Ephedraceae). These plants produce (-)-ephedrine, (+)-pseudoephedrine, (-)-norephedrine, (+)-norpseudoephedrine, (-)-N-methylephedrine, and (+)-N-methylpseudoephedrine. Racemic and (-)-ephedrine, (+)-pseudoephedrine, and (+/-)-norephedrine (phenylpropanolamine) are used clinically in the United States and are largely synthetic in origin. Current interest in Má Huáng is spurred by reports describing a "thermogenic" (calorie burning) effect provided by mixtures of ephedrine, caffeine, and aspirin. Products providing the key thermogenic compounds from natural sources are available as dietary supplements in retail outlets. Reports of potentially unsafe levels of the alkaloids, as well as possible fortification of Má Huáng-containing products with synthetic Ephedra alkaloids, prompted the development of a chiral gas chromatographic (GC) method that allows determination of alkaloid patterns and identification of isomerically impure synthetic alkaloids. Nine products were analyzed on a gamma-cyclodextrin capillary GC column. Identity of the alkaloids was verified by GC/mass spectrometry (MS) and GC/matrix isolation/Fourier transform infrared spectroscopy. No synthetic isomers were found in the dietary supplements analyzed. Three products contained only one of the ephedrine-type alkaloids. One product that listed Má Huáng as an ingredient contained no detectable ephedrine-type alkaloid. In products containing measurable quantities of these compounds, total alkaloid levels ranged from 0.3 to 56 mg/g.

Identification of a new series of fumonisins containing 3-hydroxypyridine.

A new series of fumonisins, designated FP1-3, were isolated from a culture of Fusarium moniliforme (M-2285) grown on solid corn. The new compounds contain a 3-hydroxypyridinium moiety at the C-2 position of the eicosane backbone instead of the amine found in the B series of fumonisins. The new fumonisins were characterized by UV, LC-MS-MS, 1H NMR, and 13C NMR. LC-MS analysis of culture extracts indicates that the new fumonisins can occur at levels up to approximately one-third the amount of their amine-containing analogues (FB1, FB2, and FB3).

Determination of migrants in and migration from nylon food packaging.

A method was developed to determine the amount of residual oligomers in nylon food packaging. In addition, a method was developed to measure oligomers that migrate to a food-stimulating liquid (oil) during oven cooking conditions. It was found that the total amount of nylon 6/66 oligomers that migrated from an oven baking bag to oil after heating for 30 min at 176 degrees C was 15.5 micrograms/g (ppm) or 11.9 micrograms/cm2, which represented 43% of the total amount of oligomers present in the packaging material.

Interphase cytogenetics in paraffin sections of lung tumors by non-isotopic in situ hybridization. Mapping genotype/phenotype heterogeneity.

To better understand the relationship between specific chromosome changes found in human lung tumors and their phenotypic consequences at the tissue level, an in situ hybridization procedure was optimized for use on formalin-fixed paraffin-embedded tissue sections of human lung tumors. Pretreatment heating of sections, pepsin concentration, duration of pepsin treatment, hybridization conditions, and posthybridization washing conditions were varied to determine optimum conditions. The deparaffinized sections were stained with centromeric probes for chromosomes 7 and 17, and a chromosome index for each tumor was derived by dividing the mean number of chromosome signals found on the tumor cells by the mean number of chromosome signals on normal cells (lymphocytes and fibroblasts) in the same section. This chromosome index was then compared with the DNA index determined in an adjacent section by Feulgen staining followed by image analysis quantitation. The chromosome index correlated well with the DNA index, but in some cases, chromosome 7, or 17 in other cases, was either over- or under-represented compared with the corresponding DNA index. In addition, chromosome and DNA alterations were shown to be differentially expressed within the same tissue section, correlating with a change in tumor differentiation status. These results suggest that in situ hybridization will prove to be an important tool for determining the underlying genetic basis for tissue phenotype heterogeneity by allowing genetic determinations to be made on paraffin-embedded tissue sections where tumor histological architecture is preserved.

Voice therapy techniques adapted to treatment of habit cough: a pilot study.

Patients with long-standing habit cough having no organic basis can be successfully treated with a combination of psychotherapy and speech therapy. Techniques for speech therapy are adapted from those used with hyperfunctional voice disorders to fit this debilitating laryngeal disorder.

Embryotoxic effects of benzo[a]pyrene, chrysene, and 7,12-dimethylbenz[a]anthracene in petroleum hydrocarbon mixtures in mallard ducks.

Studies with different avian species have revealed that surface applications of microliter amounts of some crude and fuel oils that coat less than 10% of the egg surface result in considerable reduction in hatching with teratogenicity and stunted growth. Other studies have shown that the embryotoxicity is dependent on the aromatic hydrocarbon content, further suggesting that the toxicity is due to causes other than asphyxia. In the present study the effects of three polycyclic aromatic hydrocarbons identified in petroleum were examined on mallard (Anas platyrhynchos) embryo development. Addition of benzo[a]pyrene (BaP), chrysene, or 7,12-dimethylbenz[a]anthracene (DMBA) to a synthetic petroleum hydrocarbon mixture of known composition and relatively low embryotoxicity resulted in embryotoxicity that was enhanced or equal to that of crude oil when 10 microliter was applied externally to eggs at 72 h of development. The order of ability to enhance embryotoxicity was DMBA greater than BaP greater than chrysene. The temporal pattern of embryonic death was similar to that reported after exposure to crude oil, with additional mortality occurring after outgrowth of the chorioallantois. Retarded growth, as reflected by embryonic body weight, crown-rump length, and bill length, was accompanied by teratogenicity. Abnormal embryos exhibited extreme stunting; eye, brain, and bill defects; and incomplete ossification. Gas chromatographic-mass spectral analysis of externally treated eggs showed the passage of aromatic hydrocarbons including chrysene through the shell and shell membranes to the developing embryos. These findings suggest that the presence of polycyclic aromatic hydrocarbons in petroleum, including BaP, chrysene, and DMBA, significantly enhances the overall embryotoxicity in avian species.

Organochlorine pollutants in small cetaceans from the Pacific and south Atlantic Oceans, November 1968-June 1976.

Organochlorine residues were analyzed in blubber, brain, or muscle tissues of 69 individuals representing 10 species of small cetaceans. Collections were made from November 1968 through June 1976 at localities in the Eastern Tropical Pacific and along the coasts of California, Hawaii, Japan, and Uruguay, Relations of residue concentrations between tissues are described for DDE and PCBs in two dolphin species. sigma DDT and PCB residues in blubber of most of the 19 individuals of the five southern California species sampled exceed concentrations that are associated with reproductive impairment in pinnipeds, although the nature of such associations is not well defined. The sigma DDT residue of 2,695 ppm in blubber of one California coastal Tursiops truncatus is one of the highest concentrations reported in tissues of members of any population of wild mammals. Except for one rough-toothed dolphin (Steno bredanensis) from Maui, Hawaii, all individuals from all localities surveyed were contaminated with organochlorine compounds. Seventeen different organochlorines were detected; greatest diversity occurred near Japan and California. This is the first report of several of these compounds in tissues of any species of marine mammals. The o,p'-isomers and metabolites of DDT were detected unusually frequently. Ratios of p,p'-DDT to p,p'-DDE in blubber of cetaceans from waters off countries where use of this pesticide has been relatively recent and ongoing were at least an order of magnitude higher than in cetaceans from United States waters.

A characterization of mitochondrial and mycoplasmal DNAs associated with cloned HeLa thymidine kinase minus cells.

HeLa S3 cells were cloned, recloned, and then selected for growth in the presence of increasing concentrations of bromodeoxyuridine. Cultures of these cloned thymidine kinase minus (TK-) cells were found to harbor mycoplasma which sedimented with mitochondria in sucrose density step gradients. Examination of mitochondrial DNA (mitDNA) components by restriction enzyme analysis and electron microscopy revealed no gross alterations in size, sequence arrangements, or replicative forms compared with mitDNA of HeLa S3 cells. Restriction enzyme cleavage sites for BamHI (one site), PstI (two sites) and HpaI (three sites) were mapped on this genome relative to the three cleavage sites for each of EcoRI and HindIII, respectively. Analysis of topological complexity revealed similar frequencies of catenated mitDNA molecules in both cloned TK- (22.5 +/- 1.5% of mass) and HeLa S3 cells )25.6 +/- 1.5% of mass). Unicircular dimers comprise 6.7 +/- 0.9% of the mitDNA mass in cloned TK- cells but were not detected in HeLa S3 mitDNA. Examination of the mycoplasmal contaminant of mitochondrial DNA after digestion with various restriction enzymes and agarose gel electrophoresis revealed that most of the DNA was distributed in discretely sized fragments in patterns that can probably be used to unambiguously identify and classify the organism.

Quantification of petroleum-type hydrocarbons in avian tissue.

Methods were developed for the analysis of 16 hydrocarbons in avian tissue. Mechanical extraction with pentane was followed by clean-up on Florisil and Silicar. Residues were determined by gas-liquid chromatography and gas-liquid, chromatography-mass spectrometry. The method was applied to the analysis of liver, kidney, fat, and brain tissue of mallard ducks (Anas platyrhynchos) fed a mixture of hydrocarbons. Measurable concentrations of all compounds analyzed were present in all tissues except brain. Highest concentrations were in fat.

Gas-liquid chromatographic determination of kepone in field-collected avian tissues and eggs.

A procedure is described for determining Kepone (decachlorooctahydro-1,3,4-metheno-2H-cyclobuta [cd] pentalene-2-one) residues in avian egg, liver, and tissue. Samples were extracted with benzene-isopropanol, and the extract was cleaned up with fuming H2SO4-concentrated H2SO4. Kepone was separated from organochlorine pesticides and polychlorinated biphenyls on a Florisil column and analyzed by electron capture gas-liquid chromatography (GLC). The average recovery from spiked tissues was 86%. The analyses performed on 14 bald eagle carcasses and livers, 3 bald eagle eggs, and 14 osprey eggs show measurable levels which indicate that Kepone accumulates in the tissues of fish-eating birds. Residues were confirmed by GLC-mass spectrometry.