Joanna L. Schroeder

Quantitative characterization of polychlorinated biphenyl mixtures (aroclors® 1248, 1254 and 1269) by gas chromatograpy using capillary columns

Abstract The polychlorinated biphenyl (PCB) compositions of Aroclors 1248, 1254 and 1260 have been determined using gas chromatography. A highly efficient glass capillary coated with teh moderately polar Dexsil 410 served as the primary column. Pairs of isomers not resolved on Dexsil 410 were easily distiguished on short, less efficient capillaries coated with Silar 5c, Apiezon L, or OV-25. The advantages associated with the high selectivity of moderately polar columns outweighed the high column efficiencies associated with non-polar phases, permitting not-very-efficient columns to provide the needed information. Meaningful quantitation was obtained through the use of the hydrogen flame ionization detector. These Aroclors, now characterized in terms of the relative molar percentages of approximately 100 different PCBs. can be used as secondary standards to obtain relative molar responses for electron capture detectors. This will permit valid application of quantitative gas- liquid chromatography to environmental samples.

Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.

Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes

By applying two different thiobarbiturate assay procedures in parallel to aliquots of a microsomal incubation mixture one can simultaneously monitor free malondialdehyde and malondialdehyde plus labile lipid peroxidation products. The levels of malondialdehyde increase continuously during the incubation of microsomes, NADPH and ferrous-ADP complex, while the lipid precursors of MDA stop forming when the system becomes depleted in NADPH. In contrast to systems in which lipids are undergoing autooxidation, NADPH-dependent lipid peroxidation does not appear to generate significant amounts of water-soluble malondialdehyde precursors. As a result, quantitative interpretation of results is straightforward in the microsomal system. In spite of the lack of specificity of the thiobarbiturate coupling reaction, interferences can be easily compensated for by using zero time controls.

PHOTOCHEMICAL REACTIONS AND PHOTOTOXICITY OF STEROLS : NOVEL SELF-PERPETUATING MECHANISM FOR LIPID PHOTOOXIDATION

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22‐ergostatetraen‐3bT‐ol (9‐DHE) and 5,7,9(11)‐cholestatrien‐3bT‐ol (9‐DDHC), respectively. Human skin lipids are known to contain 9‐DHE. We have also found 9‐DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7‐dienes (ergosterol or 7‐dehydrocholesterol), these photoactive sterols constitute a self‐regenerating source of singlet molecular oxygen (1O2) during irradiation in vivo or in vitro with UVA bT15‐400 nm). The quantum yield for photosensitization of 1O2 by 9‐DHE was estimated to be 0.09. The 1O2 is scavenged by the dienes and the rate constant for 1O2 quenching by ergosterol was found to be 1.2 times 107M‐1 s‐1 in methyl t‐butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8‐endo‐peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9‐trienes and trapped with the spin trap 5,5‐dimeth‐yl‐1‐pyrroline W‐oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.

Generation of hydrogen peroxide by incidental metal ion-catalyzed autooxidation of glutathione

Autooxidation of reduced glutathione in 50 mM buffer at pH 7.9 is indetectably slow in the presence of 1 mM DETAPAC, EDTA, TET, or tripyridine, but passing buffer through Chelex resin was insufficient to remove traces of catalytically active metals. Production of hydrogen peroxide during glutathione autooxidation was catalyzed by traces of Fe+2 or Cu+2, and to a much lesser extent by Cu+1 and Ni+2, but not to a detectable extent by Na+1, K+1, Fe+3, Al+3, Cd+2, Zn+2, Ca+2, Mg+2, Mn+2, or Hg+2. Cysteine was a much better precursor for hydrogen peroxide production than were cysteine sulfinic or sulfonic acids. The chelators EGTA, NTA, bipyridine, dimethyl glyoxime, salicylate, and Desferal were ineffective at preventing autooxidation. EDDA and 8-hydroxyquinoline were partially effective. Catalase could completely prevent the accumulation of detectable H2O2, but superoxide dismutase was only slightly inhibitory. Hydroxyl radical and singlet oxygen quenching agents (mannitol and histidine) stimulated. A mechanism for the production of H2O2 during trace metal catalyzed oxidation of glutathione is proposed, involving glutathione-complexed metal and dissolved oxygen. Although a radical intermediate can not be ruled out, no radical initiated chain reaction is necessary.

Activation of nonspecific lipase (EC 3.1.1.-) by bile salts

The enzyme nonspecific lipase (EC 3.1.1.-) from rat pancreas has been isolated and its amino acid composition determined. The amino acid composition confirms more indirect evidence that nonspecific lipase is not the same enzyme as cholesteryl ester hydrolase. Activation of the enzymatic activity by bile salts has been studied by equilibrium dialysis, gel filtration, light scattering, circular dichroism and fluorescence polarization. The binding of bile salt by the enzyme is saturable and is associated with a conformational change. Upon binding cholate, the protein experiences a decrease in beta-structure with no significant change in alpha-helix content, an increase in apparent Stokes radius, a decrease in light scattering properties, and a slight decrease in polarization of the intrinsic tryptophan fluorescence. Attachment of bile salt is associated with decreased reactivity of essential sulfhydryl groups, but no detectable change in reactivity of amino groups. A change to a more nearly spherical shape upon binding bile salt would be consistent with the experimental observations, but the exact sites of binding remain uncertain.

Chromatographic separation and quantitative determination of the metabolites of di-(2-ethylhexyl) phthalate from urine of laboratory animals

Free, glycine-conjugated, and glucuronide-conjugated metabolites of di-(2-ethylhexyl) phthalate may be stripped from urine with XAD-2 resin, derivatized, and quantitatively analyzed by liquid chromatography on a nitrile column with UV and/or radioactivity monitors. One class of metabolites requires reversed-phase chromatography or gas-liquid chromatography for its resolution. Relative molar responses of the hydrogen flame-ionization detector to these metabolites have been determined. Packed gas chromatography columns (OV-3, OV-210, cyclohexanedimethanol succinate) and fused-silica capillary columns (SP2100 and FFAP) are useful for quantitative analysis under appropriate conditions. The simplest gas chromatographic procedure permitting complete quantitative analysis requires hydrolysis of conjugates, formation of methyl esters of carboxyl groups, butyration of hydroxyl groups and chromatography on OV-3. Typical distributions of di-(2-ethylhexyl) phthalate metabolites in urine from mice, hamsters, and guinea pigs are presented.

Lipids of the earthwormLumbricus terrestris

The lipid composition of the earthwormLumbricus terrestris has been reexamined under conditions intended to avoid enzymatic and chemical alterations during storage, extraction, and fractionation procedures. The simple lipids included aliphatic hydrocarbons, steryl esters, glycerides, and at least nine different sterols, all though to be derived from the diet. Free fatty acids, previously considered to be major components of worm lipids, comprised only 0.3% of the total lipid weight. Phospholipids included (in order of relative abundance) phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, as well as sphingomyelin. Glycolipids included cerebrosides and sulfatides containing both glucose and galactose, and gangliosides containing glucosamine and sialic acid. The fatty acid compositions of these lipid classes appeared to be a mixture of what are considered typical plant, bacterial, and animal acids. Several fatty acids found in the worms, includingcis-vaccenic and eicosapentaenoic acids, were essentially absent from the dietary components, and it is concluded that these acids were synthesized in the worms. The earthworm derives much of its lipid adventitiously, but exerts at least some control over its tissue lipid composition.

DOUBLY ALLYLIC HYDROPEROXIDE FORMED IN THE REACTION BETWEEN STEROL 5,7‐DIENES AND SINGLET OXYGEN

Abstract Ergosterol and 7‐dehydrocholesterol, common 5,7‐conjugated diene sterols, react with photochemically produced singlet oxygen very efficiently to yield, in parallel pathways, the corresponding 5,8‐endoperoxides and the 7β‐hydroperoxy‐5,8(9),22‐trienol or ‐5, 8(9)‐dienol, respectively. The hydroperoxides decompose in an acid‐catalyzed reaction to generate hydrogen peroxide and the 5, 7, 9(1 1), 22‐tetraenol or 5, 7, 9(11) trienol, respectively, with 1:l stochiometry. The molar ratio of endoperoxide to hydroperoxide was constant (16:5) with two different reaction solvents, two different photosensitizers, and at all time points between 5 min and 3 h from the start of irradiation. Ergosterol did not react with either hydrogen peroxide or superoxide ion under our reaction conditions. Inhibition studies with nitrogen, 2,5‐dimethylfuran, βcarotene, and tert‐butanol confirmed the involvement of singlet oxygen in these reactions. The unstable hydroperoxide would be expected to have undesirable biological consequences if formed in vivo.

Rapid isolation of microsomes for studies of lipid peroxidation

Conventional isolation of microsomes by high-speed centrifugation from isotonic sucrose requires exposure to air for several hours, leading to the formation of low levels of lipid peroxidation products. Sucrose interferes in protein and malondialdehyde assays and provides no protection against lipid peroxidation during workup. A new procedure for the purification of microsomes from rat liver substitutes mannitol (a hydroxyl radical scavenger) for sucrose and takes advantage of the properties of morpholinopropane sulfonic acid (MOPS) buffer and triethylenetetramine to provide protection against lipid peroxidation during the rapid (less than one hour) workup and subsequent low-temperature storage. The microsomal fractions prepared by the proposed method are free of detectable mitochondrial contamination and at least as pure overall as those prepared by the conventional method, but they have higher glucose-6-phosphatase and laurate hydroxylase activities and significantly less malondialdehyde than conventional microsomes at the time isolation is complete. Laurate hydroxylase activity is more stable during frozen storage in mannitol medium. The kinetics of lipid peroxidation in vitro are quite different for microsomes prepared by the two methods.